Nitric oxide-releasing particles for nitric oxide therapeutics and biomedical applications

ABSTRACT

The presently disclosed subject matter relates to nitric oxide-releasing particles for delivering nitric oxide, and their use in biomedical and pharmaceutical applications.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/014,939, filed Aug. 30, 2013, which is a continuation of U.S. patentapplication Ser. No. 13/157,036, filed Jun. 9, 2011, which is acontinuation of U.S. patent application Ser. No. 11/887,041, filed Jan.15, 2009, now abandoned, which is a national stage application ofPCT/US2006/020781, filed May 30, 2006, which claims the benefit of U.S.Provisional Patent Application Ser. No. 60/685,578, filed May 27, 2005;the disclosure of which is incorporated herein by reference in itsentirety.

GOVERNMENT INTEREST

This invention was made with U.S. Government support from NationalInstitutes of Health Grant Number EB000708. Thus, the U.S. Governmenthas certain rights in the invention.

TECHNICAL FIELD

The presently disclosed subject matter provides nitric oxide-releasingparticles and their use in biomedical and pharmaceutical applications.More particularly, in some embodiments, the presently disclosed subjectmatter provides particles that release nitric oxide in a controlled andtargeted manner, thereby prolonging the therapeutic effects of nitricoxide and improving the specificity of nitric oxide delivery to targetedcells and/or tissues.

ABBREVIATIONS

-   -   AFM=atomic force microscopy    -   AEAP3=N-(6-aminoethyl)-aminopropyltrimethoxysilane    -   AEMP3=(aminoethylaminomethyl)-phenethyl trimethoxysilane    -   AHAP3=N-(6-aminohexyl)-aminopropyltrimethoxysilane    -   AIBN=a,a′-azobisisobutyronitrile    -   atm=atmosphere    -   BSA=bovine serum albumin    -   ° C.=degrees Celsius    -   CFU=colony forming units    -   CP/MAS=cross polarization/magic angle spinning    -   CTAB=cetyltrimethyl ammonium bromide    -   DET3=N-[3-(trimethyoxysilyl)propyl]-diethylenetriamine    -   EtOH=ethanol    -   FA=folic acid    -   FITC=fluorescein isothiocyanate    -   g=grams    -   GOx=glucose oxidase    -   h=hours    -   HPU=hydrophilic polyurethane    -   MAP3=methylaminopropyl trimethoxysilane    -   MeOH=methanol    -   mg=microgram    -   μm=micrometers    -   min=minutes    -   mL=milliliter    -   mol %=mole percent    -   MPC=monolayer protected cluster    -   MRI=magnetic resonance imaging    -   MTMOS=methyltrimethoxysilane    -   nA=nanoampere    -   NaOMe=sodium methoxide    -   nm=nanometer    -   NMR=nuclear magnetic resonance    -   NO=nitric oxide    -   [NO]_(m)=maximum flux of nitric oxide release    -   O₃=ozone    -   OD=optical density    -   PAMA=polyamidoamine    -   M    -   pmol=picomole    -   ppb=parts-per-billion    -   PPl=polypropylenimine    -   ppm=parts-per-million    -   TPU=TECOFLEX® polyurethane    -   TEM=transmission electron microscopy    -   TEOS=tetraethyl orthosilicate    -   TGA=thermal gravimetric analysis    -   TMOS=tetramethyl orthosilicate    -   TMRM=tetramethylhodamine    -   t[NO]=total amount of nitric oxide    -   UV=ultraviolet    -   Vis=visible

BACKGROUND

The discovery of the multifaceted role of nitric oxide (NO) in biology,physiology, and pathophysiology, see Marietta, M. A., at al.,BioFactors, 2, 219-225 (1990), has led to the search for nitric oxidedonors capable of controlled nitric oxide release. See Keefer, L. K.,Chemtech, 28, 30-35 (1998). To date, researchers have discovered that NOregulates a range of biological processes in the cardiovascular,gastrointestinal, genitourinary, respiratory, and central and peripheralnervous systems. See Ignarro, L. J., Nitric Oxide: Biology andPathobiology; Academic Press: San Diego, 2000; and Ignarro, L. J. etal., Proc. Natl. Acad. Sci., U.S.A., 84, 9265-9269 (1987). Furthermore,the discovery of NO as a vasodilator and its identification as both anantibiotic and a tumoricidal factor have made NO an attractivepharmaceutical candidate. See, for example, Radomski, M. W., et al., Br.J. Pharmacol., 92, 639-646 (1987); Albina, J. E., and Reichner, J. S.;Canc. Metas. Rev., 17, 19-53 (1998); Nablo, B. J., at al., J. Am. Chem.Soc., 123, 9712-9713 (2001); Cobbs, C. S., et al., Cancer Res., 55,727-730 (1995); Jenkins, D. C., at al., Proc. Natl. Acad. Sci., U.S.A.,92, 4392-4396 (1995); and Thomsen, L. L., et al., Br. J. Cancer., 72,41-44 (1995).

Several nitric oxide donors have been reported, the most notable beingN-diazeniumdiolates. Generally, N-diazeniumdiolate NO donors are smallmolecules synthesized by the reaction of amines with NO at elevatedpressure and have been used, for example, to spontaneously generate NOin aqueous solution. See Hrabie, J. A. and Keefer, L. K., Chem. Rev.,102, 1135-1154 (2002).

Therapeutic strategies to explore the activities of nitric oxide donors,for example, to kill tumor cells, are problematic in part because thenitric oxide delivery systems known in the art release or donate nitricoxide indiscriminately. Thus, there is a need in the art for a nitricoxide delivery system that releases or donates nitric oxide in acontrolled and/or targeted manner to facilitate an improvedunderstanding of the function of NO in physiology and to provide for thedevelopment of NO-associated therapies.

SUMMARY

In some embodiments, the presently disclosed subject matter provides anitric oxide (NO)-releasing particle, comprising a nitric oxide donor,an exterior region, and an interior region having a volume, the volumeof the interior region at least partially filled by a core selected fromthe group consisting of:

-   -   (a) a metallic cluster;    -   (b) a dentritic network;    -   (c) a co-condensed silica network; and    -   (d) a combination thereof.

In some embodiments, the interior region further comprises an organiclinker selected from the group consisting of a labile linker responsiveto changes in pH, a labile linker sensitive to electromagneticradiation, a labile linker susceptible to degradation by enzymaticaction, a hydrophobic linker, an amphiphilic linker, and combinationsthereof.

In some embodiments, the NO donor is selected from the group consistingof a diazeniumdiolate, a nitrosamine, a hydroxylamine, a nitrosothiol, ahydroxyl amine, and a hydroxyurea. In some embodiments the NO donor iscovalently bound to one of the interior region, the exterior region, thecore, or to combinations thereof. In some embodiments the NO donor isencapsulated in one of the interior region, the exterior region, thecore, or to combinations thereof. In some embodiments the NO donor isassociated with part of the particle via a non-covalent interactionselected from the group consisting of Van der Waals interactions,electrostatic forces, hydrogen bonding, or combinations thereof.

In some embodiments, the exterior region comprises one or more chemicalmoieties selected from the group consisting of moities that modulate thenitric oxide release kinetics, affect the biocompatibility or thebiodistribution of the particle, provide for targeted delivery of theparticle, impart an ability to image or track the particle, affect thesolubility of the particle, provide a therapeutic effect, orcombinations thereof.

In some embodiments, the core is a metallic cluster further comprising acomponent selected from the group consisting of gold, platinum, silver,magnetite, a quantum dot, or combinations thereof. In some embodiments,the metallic cluster is a monolayer protected gold cluster.

In some embodiments, the core is a dendritic network selected from thegroup consisting of a polypropylenimine dendrimer, a polyamidoaminedendrimer, a polyaryl ether dendrimer, a polypeptide dendrimer, apolyester dendrimer, a polyamide dendrimer, a dendritic polyglycerol,and a triazine dendrimer. In some embodiments the dendritic network ishyperbranched.

In some embodiments, the core is a co-condensed silica networksynthesized from the condensation of a silane mixture comprising analkoxysilane and an aminoalkoxysilane. In some embodiments, thealkoxysilane is a tetraalkoxysilane of the formula Si(OR)₄, wherein R isalkyl, and the aminoalkoxysilane has a formula selected from:

-   -   (a) an aminoalkoxysilane of the formula R″—(NH—R′)_(n)—Si(OR)₃,        wherein R is alkyl, R′ is alkylene, branched alkylene, or        aralkylene, n is 1 or 2, and R″ is selected from the group        consisting of alkyl, cycloalkyl, aryl, and alkylamine;    -   (b) an aminoalkoxysilane of the formula NH[R′—Si(OR)₃]₂, wherein        R is alkyl and R′ is alkylene;    -   (c) an aminoalkoxysilane wherein the amine is substituted by a        diazeniumdioiate, said aminoalkoxysilane having the formula        R″—N(NONCYX+)-R′—Si(OR)3, wherein R is alkyl, R′ is alkylene or        aralkylene, R″ is alkyl or alkylamine, and X+ is a cation        selected from the group consisting of Na+ and K+; and    -   (d) a combination thereof.

In some embodiments the siline mixture comprises between about 10 mol %to about 99 mol % of tetraalkoxysilane and about 1 mol % to about 90 mol% of aminoalkoxysilane. In some embodiments, the silane mixture furthercomprises about 0 mol % to about 20 mol % of fluorinated silane; about 0mol % to about 20 mol % of cationic or anionic silane; and about 0 mol %to about 20 mol % of aikylsilane.

In some embodiments, the tetraalkoxysilane is selected from groupconsisting of tetramethyl orthosilicate and tetraethyl orthosilicate.

In some embodiments, the aminoalkoxysilane is selected from the groupconsisting of:

-   N-(6-aminohexyl)aminomethyltrimethoxysilane;-   N-(6-aminohexyl)aminopropyltrimethoxysilane;-   N-(6-aminoethyl)aminopropyltrimethoxysilane;-   (3-trimethoxysilylpropyl)diethylenetriamine;-   (aminoethylaminomethyl)phenethyltrimethoxysilane;-   [3-(methylamino)propyl]trimethoxysilane;-   N-butylaminopropyltrimethoxysilane;-   N-ethylaminoisobutyltrimethoxysilane;-   N-phenylaminopropyltrimethoxysilane;-   N-cyclohexylaminopropyltrimethoxysilane;-   Bis[3-(trimethoxysilyl)propyl]amine; and-   Bis[(3-trimethoxysilyl)propyl]ethylenediamine.

In some embodiments the fluorinated silane is selected from the groupconsisting of (heptadecafluoro-1,1,2,2-tetrahydrodecyl)-triethoxysilane,(3,3,3-trifluoropropyl)trimethoxysilane, and(perfluoroalkyl)ethyltriethoxysilane.

In some embodiments, the cationic or anionic silane is selected from thegroup consisting of:

-   -   N—N-didecyl-N-methyl-N-(3-trimethoxysilyl)ammonium chloride;        octadecyldimethyl(3-trimethoxysilylpropyl)ammonium chloride;        3-trihydroxysilylpropylmethyl phosphonate, sodium salt; and        carboxylethylsilanetriol, sodium salt.

In some embodiments the alkylsilane is selected from the groupconsisting of methyltrimethoxysilane, butyltrimethoxysilane,butyltriethoxysilane, propyltrimethoxysilane, andoctadecyltrimethoxysilane.

In some embodiments, the NO-releasing particle comprising a co-condensedsilica network core and the NO donor is synthesized using a“post-charging” method wherein the NO donor is formed after thecondensation of the silica network in some embodiments, the NO-releasingparticle comprising a co-condensed silica network core is synthesizedusing a “pre-charging” method wherein the NO donor is formed prior tothe condensation of the silica network.

In some embodiments, the organic linker comprises a functional groupcapable of conferring an on/off state of nitric oxide release to thenitric oxide-releasing particle, wherein the functional group isselected from the group consisting of an ester, a hydrazone, an acetal,a thiopropionate, a photolabile moiety, and an amino acid sequencesubject to enzymatic degradation.

In some embodiments, the exterior comprises a moiety capable ofdelivering the NO-releasing particle to a target. In some embodiments,the target is selected from a cell, a tissue, and an organ, In someembodiments, the cell is a cancer cell.

In some embodiments, the moiety capable of delivering the NO-releasingparticle to the target is selected from the group consisting of aprotein responsible for antibody/antigen interaction, folic acid,guanidine, transferrin, a hormone, carbohydrates, a peptide containingthe amino acid sequence RGD, and TAT peptides.

In some embodiments, the exterior comprises a moiety selected from anitric oxide donor, a (poly)ethyleneoxide, a (poly)urethane, anN-(2-hydroxypropyl) methacrylamide copolymer, lactide/glycolidecopolymers (e.g. poly(lactic-co-glycolic acid, PGA), a sugar, afluorescent organic dye, an MRI contrast agent, a thiol, amethyl-terminated alkyl chain, an antibiotic, an anti-cancertherapeutic, a sulfonate, a carboxylate, a phosphate, a cationic amine,a quaternary amine, and combinations thereof.

In some embodiments, the NO-releasing particle has a diameter of frombetween about 1 nm and about 1000 nm. In some embodiments, the particlehas a metallic cluster core and the diameter of the particle is frombetween about 1 nm and about 5 nm. In some embodiments the particle hasa co-condensed silica network core and has a diameter of between about 2nm and about 10 μm.

In some embodiments, the presently disclosed subject Matter provides amethod or a formulation for delivering nitric oxide to a subject, insome embodiments, the method comprises administering an effective amountof a NO-releasing particle to the subject, said particle comprising a NOdonor, an exterior region, and an interior region having a volume, thevolume of the interior region at least partially filled by a coreselected from:

(a) a metallic cluster;

(b) a dendritic network;

(c) a co-condensed silica network; and

(d) a combination thereof.

In some embodiments, the presently disclosed subject matter provides amethod of treating a disease state in a subject in need of treatmentthereof wherein the method comprises administering to a subject in needof treatment a NO-releasing particle comprising a NO donor, an exteriorregion, and an interior region having a volume, the volume at leastpartially filled by a core selected from:

(a) a metallic cluster;

(b) a dendritic network;

(c) a co-condensed silica network; and

(d) a combination thereof.

In some embodiments the disease state is selected from cancer, acardiovascular disease, a microbial infection, platelet aggregation andplatelet adhesion caused by the exposure of blood to a medical device,pathological conditions resulting from abnormal cell proliferation,transplantation rejections, autoimmune diseases, inflammation, vasculardiseases; scar tissue, wound contraction, restenosis, pain, fever,gastrointestinal disorders, respiratory disorders, sexual dysfunctions,and sexually transmitted diseases.

In some embodiments, the presently disclosed subject matter providespolymeric films containing NO-releasing particles. In some embodimentsthe polymeric films can be used to coat medical devices. In someembodiments, the medical device is one of an arterial stent, a guidewire, a catheter, a trocar needle, a bone anchor, a bone screw, aprotective plating, a hip or joint replacement, an electrical lead, abiosensor, a probe, a suture, a surgical drape, a wound dressing, and abandage.

In some embodiments, the presently disclosed subject matter provides adetergent comprising a NO-releasing particle.

Thus, it is an object of the presently disclosed subject matter toprovide nitric oxide-releasing particles. It is another object of thepresently disclosed subject matter to provide nitric oxide-releasingparticles for the targeted delivery of nitric oxide to a specific celland/or tissue. It is another object of the presently disclosed subjectmatter to provide the ability to trigger the release of nitric oxidefrom nitric oxide-releasing particles.

Certain objects of the presently disclosed subject matter having beenstated herein above, which are addressed in whole or in part by thepresently disclosed subject matter, other objects and aspects willbecome evident as the description proceeds when taken in connection withthe accompanying Examples as best described herein below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a nitric oxide (NO)-releasingparticle comprising a core CR, an interior region IR, a linker LK havinga labile portion LP, a nitric oxide (NO) donor NO and an exterior EP.

FIG. 2 is a synthesis scheme for preparing the presently disclosedNO-releasing monolayer protected cluster (MPC) gold nanoparticles.

FIG. 3 is a schematic representation of the synthesis of NO-releasingparticles via the co-condensation of silica networks from mixtures ofalkoxysilanes and aminoalkoxysilanes followed by treatment of theco-condensed silica network with NO gas.

FIG. 4A is a schematic representation of the extent of NO donordistribution in N-diazeniumdiolate (darker spheres)-modified silicaparticles synthesized by a surface grafting method.

FIG. 4B is a schematic representation of the extent of NO donordistribution in N-diazeniumdiolate (darker spheres)-modified silicaparticles synthesized by “one-pot” co-condensation of silica networksfrom silane mixtures comprising alkoxysilanes and aminoalkoxysilanes.

FIG. 5A is a schematic representation of the synthesis of NO-releasingco-condensed silica particles using a “post-charging” method, whereinamino groups from aminoalkoxysilanes are reacted with NO gas afterco-condensation in a silica network.

FIG. 5B is a schematic representation of the synthesis of NO-releasingco-condensed silica particles using a “pre-charging” method, whereinaminoalkoxysilanes are reacted with NO gas prior to co-condensation toform a silica network.

FIG. 6A is the structure ofN-(2-aminoethyl)-3-aminopropyltrimethoxysilane (AEAP3).

FIG. 6B is the structure of(aminoethylaminomethyl)phenethyltrimethoxysilane (AEMP3).

FIG. 6C is the structure of N-(6-aminohexypaminopropyltrimethoxysilane(AHAP3).

FIG. 6D is the structure ofN43-(trimethoxysilyl)propyl]diethylenetriarnine (DET3).

FIG. 7 is a schematic representation of the templated synthesis ofmesoporous co-condensed silica networks using micelles as templatingagents to direct pore formation.

FIG. 8 is a schematic representation of a portion of the NO-releasingparticle as previously described for FIG. 1, further showing that thelabile portion LP of the linker LK can be positioned at varyingdistances from the particle exterior EP. Position A is farthest awayfrom the exterior, position B is located in the middle of the linker,and position C is closest to the exterior of the particle.

FIG. 9A shows a generalized structure of a medically segmentedpolyurethane. Soft units are represented by the shaded ellipses, hardunits by shaded rectangles, and expander units by shaded circles.

FIG. 9B shows the structure of TECOFLEX® polyurethane, wherein n and n′are integers.

FIG. 10A is a ¹H NMR spectrum of hexanethiol-functionalized goldnanoparticles.

FIG. 10B is a ¹H NMR spectrum of bromine-functionalized goldnanoparticles, The —CH₂Br peaks appear at 3.4 ppm.

FIG. 10C is a ¹H NMR spectrum of ethylenediamine-functionalized goldnanoparticles. The —CH₂NH peaks appear from 2.5 to 3.0 ppm.

FIG. 11 is a scheme for a two-step synthesis of11-bromo-1-undecanethiol.

FIG. 12 is a schematic representation of an analytical method formeasuring nitric oxide.

FIG. 13 is a plot showing nitric oxide release profiles from monolayer,protected cluster (MPC) gold nanoparticles derivatized with variousdiamines. Line a is the nitric oxide release profile of underivatizedMPC gold nanoparticles. Line b is the nitric oxide release profile fromMPC gold nanoparticles derivatized with 14% ethylenediamine. Line cshows the nitric oxide release profile from MPC gold nanoparticlesderivatized with 21% ethylenediamine. Line d shows the nitric oxiderelease profile from MPC gold nanoparticles derivatized with 21%ethylenediamine. Line e shows the nitric oxide release profile from MPCgold nanoparticles derivatized with 21% diethylenetriamine. Line f showsthe nitric oxide release profile from MPC gold nanoparticles derivatizedwith 21% hexanediamine.

FIG. 14 is a schematic representation showing the release of nitricoxide from functionalized monolayer protected cluster (MPG) goldnanoparticles.

FIG. 15 is a schematic representation of the chemical structure ofpolypropylenimine hexadecaamine dendrimer (DAB-Am-16).

FIG. 16 is a schematic representation of the chemical structure ofpolypropylenimine tetrahexacontaamine dendrimer (DAB-Am-64).

FIG. 17 is a graph showing the nitric oxide release profile forDAB-C7-16 NaOMe/MeOH.

FIG. 18 is a graph showing the nitric oxide release profile forDAB-C7-64 NaOMe/MeOH.

FIG. 19 is a synthesis route to NO-releasing silica particles accordingto the method described by Zhand, H., et al., J. Am. Chem. Soc., 125,5015 (2003).

FIG. 20A is a contact mode atomic force microscope (AFM) image ofcontrol silica (TEOS only).

FIG. 20B is a contact mode atomic force microscope (AFM) image of silicawith 10 mol % of AHAP3 (balance TEOS).

FIG. 20C is an enlargement of the atomic force microscope (AFM) imagefrom FIG. 29B showing a single particle.

FIG. 20D is a contact mode atomic force microscope (AFM) image of 10 mol% AEAP3.

FIG. 20E is a contact mode atomic force microscope (AFM) image of 17 mol% AEAP3 silica particles on a mica surface.

FIG. 20F is a graph showing the relationship between the AEAP3 contentin the silica composite and the resulting particle size.

FIG. 21A is a plot showing the solid-state ²⁹S1 cross polarization/magicangle spinning (CP/MAS) NMR spectra of co-condensed silica with variousamounts of AEAP3: (a) 0% AEAP3 (control), (b) 10 mol % AEAP3 (balanceTEOS), (c) 13 mol % AEAP3 (balance TEOS); and 17 mol % AEAP3 (balanceTEOS).

FIG. 21B is a schematic showing the structures related to the siliconchemical environments at the surface of AEAP3-modified silicacomposites.

FIG. 21C is a plot of % surface coverage of co-condensed amine ligandsversus AEAP3 content loaded during the synthesis of AEAP3-modifiedsilica composites.

FIG. 22 is a NO-release profile of NO release from co-condensed silicacontaining 10 mol % AHAP3 (dashed line) and 17 mol % AEAP3 (solid line).The inset shows a plot of total NO-release over time of the same twosilica types.

FIG. 23 is a plot of NO release of co-condensed silica nanoparticlescontaining AEAP3 as a function of pH at 37° C. The inset is a plot oftotal NO release.

FIG. 24A is a schematic representation showing a cross section of amesoporous NO-releasing silica particle prepared by a templatedsynthesis using the surfactant cetyltrimethyl ammonium bromide (CTAB) asa template. The shaded area represents co-condensed silica network,while the small shaded circles represent NO-donors in the co-condensedsilica network. The unshaded area represents pores in the particleformed from the removal of the CTAB template after the silanecondensation reaction.

FIG. 24B is a contact mode atomic force microscope (AFM) image of amesoporous N-(6-aminoethyl)aminopropyltrimethoxysilane (AEAP3)-silicaparticle prepared using cetyltrimethyl ammonium bromide (CTAB) as atemplate.

FIG. 25 is a plot showing the nitric oxide release profile of mesoporousN-(6-aminoethyl)aminopropyltrimethoxysilane (AEAP3)-silica (3 mg ofparticles in phosphate buffer solution (PBS) at 37° C.).

FIG. 26 is a graph of the cytotoxicity of control (dark circles) andNO-releasing silica prepared with 45 mol % AHAP3 (open circles) onovarian epithelial tumor cells.

FIG. 27 is a graph showing the cytotoxicity of control (dark squares)and NO-releasing MAP3 co-condensed (open squares) silica nanoparticleson normal (T29, immortalized) cells as well as the cytotoxicity ofcontrol (dark circles) and NO-releasing MAP3 co-condensed (open circles)silica nanoparticles on tumor (A2780) cells.

FIG. 28 is a bar graph showing the effect of silica particle size (75mol % MAP3, balance TEOS) on cytotoxicity against normal T29 (shadedbars) and tumor A2780 (striped bars) cell lines. P<0.001 compared withcontrol MAP3-treated group. Control MAP3 silica (indicated by thebrackets) are non NO-releasing and have a diameter of 80 nm, S-MP3silica has a diameter of 80 nm, L-MAP3 silica has a diameter of 350 nm.

FIG. 29A is a laser spanning microscope image of A2780 ovarian cancercells taken at 5 min after incubation with FITC-labeled MAP3 silicananoparticles.

FIG. 29B is a laser scanning microscope image of A2780 ovarian cancercells taken at 60 min after incubation with FITC-labeled MAP3 silicananoparticles.

FIG. 29C is a laser scanning microscope image of A2780 ovarian cancercells taken at 5 min after incubation with 100 nm tetramethylrhodamine(TMRM) mitochondrial stain.

FIG. 29D is a laser scanning microscope image of A2780 ovarian cancercells taken at 60 min after incubation with 100 nm tetramethylrhodamine(TMRM) mitochondrial stain.

FIG. 30A is a photograph showing colonies of P. aeruginosa that formedon nutrient agar plates after incubation with sterile phosphate bufferedsolution (PBS) at 37° C.

FIG. 30B is a photograph showing colonies of P. aeruginosa that formedon nutrient agar plates after incubation with control (non NO-releasing)AEAP3 silica nanoparticles at 37° C.

FIG. 30C is a photograph showing colonies of P. aeruginosa that formedon nutrient agar plates after incubation with NO-releasing 45 mol %AEAP3 silica nanoparticles at 37° C.

FIG. 31 is a plot showing the in vitro bactericidal activity ofNO-releasing silica nanoparticles (45 mol % AEAP3, balance TEOS) againstP. aeruginosa as a function of nanoparticle concentration.

FIG. 32 is a schematic representation of the synthesis of a NO-releasingnanoparticle having a magnetite core covered by a layer of co-condensedsilica containing amine groups that can form NO-donors.

FIG. 33 is an atomic force microscope (AFM) image ofmagnetite/N-(6-aminohexypaminopropyltrimethoxysilane (AHAP3, 10 mol%)-functionalized silica particles.

FIG. 34 is a graph showing the NO release profile of magnetite/silicacore particles (lower line) compared to the NO-release profile ofparticles having cores of the same silica composition without magnetite(upper line).

The inset is a graph of total NO release.

FIG. 35A is a phase contrast optical micrograph showing P. aeruginosaadhesion (dark areas) to a control film (a non NO-releasingpolyurethane). Magnification=5×.

FIG. 35B is a phase contrast optical micrograph showing P. aeruginosaadhesion (dark areas) to a NO-releasing particle-containing film.Magnification=5×.

FIG. 36 is a schematic representation of the structure for a Ptelectrode of a glucose sensor having a NO-releasing layer formed from apolymeric film comprising NO-releasing co-condensed silicananoparticles.

DETAILED DESCRIPTION

The presently disclosed subject matter will now be described more fullyhereinafter with reference to the accompanying Examples, in whichrepresentative embodiments are shown. The presently disclosed subjectmatter can, however, be embodied in different forms and should not beconstrued as limited to the embodiments set forth herein. Rather, theseembodiments are provided so that this disclosure will be thorough andcomplete, and will fully convey the scope of the embodiments to thoseskilled in the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this presently described subject matter belongs. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

Throughout the specification and claims, a given chemical formula orname shall encompass all optical and stereoisomers, as well as racemicmixtures where such isomers and mixtures exist.

I. DEFINITIONS

Following long-standing patent law convention, the terms “a” and “an”mean one or more when used in this application, including the claims.

The term “amphipathic” as used herein refers to a chemical moiety havinga hydrophobic region and a hydrophilic region.

The term “cancer” as used herein refers to diseases caused byuncontrolled cell division and the ability of cells to metastasize, orto establish new growth in additional sites. The terms “malignant”,“malignancy”, “neoplasm”, “tumor” and variations thereof refer tocancerous cells or groups of cancerous cells.

Specific types of cancer include, but are not limited to, skin cancers,connective tissue cancers, adipose cancers, breast cancers, lungcancers, stomach cancers, pancreatic cancers, ovarian cancers, cervicalcancers, uterine cancers, anogenital cancers, kidney cancers, bladdercancers, colon cancers, prostate cancers, central nervous system (CNS)cancers, retinal cancer, blood cancers, and lymphoid cancers.

As used herein, the term “electromagnetic radiation” refers to electricand magnetic waves such as, but not limited to, gamma rays, x-rays,ultraviolet light, visible light, infrared light, microwaves, radar andradio waves.

The term “hydrophobic” refers to a chemical compound or moiety that, toa given extent, repels or does not interact with water throughnon-covalent forces such as hydrogen bonding or electrostaticinteractions. A compound can be strongly hydrophobic or slightlyhydrophobic. The calculated dielectric constant of a compound or groupcan be used to predict the level or degree of hydrophobicity of thecompound or moiety. Compounds or moieties with lower dielectricconstants will be more hydrophobic. In particular, a “hydrophobiclinker” is one that will protect a labile linker or a NO donor in aNO-releasing particle from exposure to water when the particle is placedin an aqueous environment for a period of time. A more hydrophobiclinker will protect a NO donor or labile linker from water for a longerperiod of time.

The term “hydrophilic” refers to a compound or moiety that will interactwith water to given extent.

The term “ionizable” refers to a group that is electronically neutral(La, uncharged) in a particular chemical environment (e.g., at aparticular pH), but that can be ionized and thus rendered positively ornegatively charged in another chemical environment (e.g., at a higher orlower pH).

The term “mesoporous” as used herein refers to an object, such as aparticle, comprising pores in the range of between about 20-500angstroms.

The term “metallic” refers to metals, metal alloys, metal salts, andmetal oxides. Thus, the term metallic refers to particles comprisingmetal ions, such as, but not limited to, gold, silver, copper, platinum,and titanium, as well as semiconductor particles and magnetic particles(e.g., particles comprising iron oxides).

The terms “semiconductor nanocrystal” and “quantum dot” are usedinterchangeably herein to refer to semiconductor nanoparticlescomprising an inorganic crystalline material that is luminescent (i.e.,that is capable of emitting electromagnetic radiation upon excitation),and including an inner core of one or more first semiconductor materialsthat is optionally contained within an overcoating or “shell” of asecond semiconductor material. A semiconductor nanocrystal coresurrounded by a semiconductor shell is referred to as a “core/shell”semiconductor nanocrystal. The surrounding shell material can optionallyhave a bandgap energy that is larger than the bandgap energy of the corematerial and can be chosen to have an atomic spacing close to that ofthe core substrate.

Suitable semiconductor materials for the core and/or shell include, butare not limited to, materials comprising a first element selected fromGroups 2 and 12 of the Periodic Table of the Elements and a secondelement selected from Group 16. Such materials include, but are notlimited to ZnS, ZnSe, ZnTe, CDs, CdSe, CdTe, HgS, HgSe, HgTe, MgS, MgSe,MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, and the like.Suitable semiconductor materials also include materials comprising afirst element selected from Group 13 of the Periodic Table of theElements and a second element selected from Group 15. Such materialsinclude, but are not limited to, GaN, GaP, GaAs, GaSb, InN, inP, InAs,InSb, and the like. Semiconductor materials further include materialscomprising a Group 14 element (Ge, Si, and the like); materials such asPbS, PbSe and the like; and alloys and mixtures thereof. As used herein,all reference to the Periodic Table of the Elements and groups thereofis to the new IUPAC system for numbering element groups, as set forth inthe Handbook of Chemistry and Physics, 81st Edition (CRC Press, 2000).

By “luminescence” is meant the process of emitting electromagneticradiation (light) from an object. Luminescence results when a systemundergoes a transition from an excited state to a lower energy statewith a corresponding release of energy in the form of a photon. Theseenergy states can be electronic, vibrational, rotational, or anycombination thereof. The transition responsible for luminescence can bestimulated through the release of energy stored in the system chemicallyor added to the system from an external source. The external source ofenergy can be of a variety of types including chemical, thermal,electrical, magnetic, electromagnetic, and physical, or any other typeof energy source capable of causing a system to be excited into a statehigher in energy than the ground state. For example, a system can beexcited by absorbing a photon of light, by being placed in an electricalfield, or through a chemical oxidation-reduction reaction. The energy ofthe photons emitted during luminescence can be in a range fromlow-energy microwave radiation to high-energy x-ray radiation.Typically, luminescence refers to photons in the range from UV to IRradiation.

The term “fluorescent” refers to a compound or chemical group that emitslight following exposure to electromagnetic radiation.

The terms “nitric oxide donor” or “NO donor” refer to species thatdonate, release and/or directly or indirectly transfer a nitric oxidespecies, and/or stimulate the endogenous production of nitric oxide invivo and/or elevate endogenous levels of nitric oxide in vivo such thatthe biological activity of the nitric oxide species is expressed at theintended site of action.

The terms “nitric oxide releasing” or “nitric oxide donating” refer tomethods of donating, releasing and/or directly or indirectlytransferring any of the three redox forms of nitrogen monoxide (NO+,NO⁻, NO). In some cases, the nitric oxide releasing or donating isaccomplished such that the biological activity of the nitrogen monoxidespecies is expressed at the intended site of action.

The term “microbial infection” as used herein refers to bacterial,fungal, viral, and yeast infections.

The term “about,” as used herein, when referring to a value or to anamount of mass, weight, time, volume, or percentage is meant toencompass variations of ±20% or ±10%, more preferably ±5%, even morepreferably ±1%, and still more preferably ±0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethod.

The “patient” or “subject” treated in the many embodiments disclosedherein is desirably a human patient, although it is to be understoodthat the principles of the presently disclosed subject matter indicatethat the presently disclosed subject matter is effective with respect toall vertebrate species, including mammals, which are intended to beincluded in the terms “subject” and “patient.” In this context, a mammalis understood to include any mammalian species in which treatment isdesirable, particularly agricultural and domestic mammalian species,such as horses, cows, pigs, dogs, and cats.

As used herein the term “alkyl” refers to C₁₋₂₀ inclusive, linear (i.e.,“straight-chain”), branched, or cyclic, saturated or at least partiallyand in some cases fully unsaturated (i.e., alkenyl and alkynyl)hydrocarbon chains, including for example, methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, octyl, ethenyl,propenyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl, propynyl,butynyl, pentynyl, hexynyl, heptynyl, and alkenyl groups. “Branched”refers to an alkyl group in which a lower alkyl group, such as methyl,ethyl or propyl, is attached to a linear alkyl chain. Exemplary branchedalkyl groups include, but are not limited to, isopropyl, isobutyl,tert-butyl, “Lower alkyl” refers to an alkyl group having 1 to about 8carbon atoms (i.e., a C₁₋₈ alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8carbon atoms. “Higher alkyl” refers to an alkyl group having about 10 toabout 20 carbon atoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or20 carbon atoms. In certain embodiments, “alkyl” refers, in particular,to C₁₋₈ straight-chain alkyls. In other embodiments, “alkyl” refers, inparticular, to C₁₋₈ branched-chain alkyls.

Alkyl groups can optionally be substituted (a “substituted alkyl”) withone or more alkyl group substituents, which can be the same ordifferent. The term “alkyl group substituent” includes but is notlimited to alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl,aryloxyl, alkoxyl, alkylthio, arylhio, aralkyloxyl, araikylthio,carboxyl, alkoxycarbonyl, oxo, and cycloalkyl. There can be optionallyinserted along the alkyl chain one or more oxygen, sulfur or substitutedor unsubstituted nitrogen atoms, wherein the nitrogen substituent ishydrogen, lower alkyl (also referred to herein as “alkylaminoalkyl”), oraryl.

Thus, as used herein, the term “substituted alkyl” includes alkylgroups, as defined herein, in which one or more atoms or functionalgroups of the alkyl group are replaced with another atom or functionalgroup, including for example, alkyl, substituted alkyl, halogen, aryl,substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino,dialkylamino, sulfate, and mercapto.

The term “aryl” is used herein to refer to an aromatic substituent thatcan be a single aromatic ring, or multiple aromatic rings that are fusedtogether, linked covalently, or linked to a common group, such as, butnot limited to, a methylene or ethylene moiety. The common linking groupalso can be a carbonyl, as in benzophenone, or oxygen, as indiphenylether, or nitrogen, as in diphenylamine. The term “aryl”specifically encompasses heterocyclic aromatic compounds. The aromaticring(s) can comprise phenyl, naphthyl, biphenyl, diphenylether,diphenylamine and benzophenone, among others. In particular embodiments,the term “aryl” means a cyclic aromatic comprising about 5 to about 10carbon atoms, e.g., 5, 6, 7, 8, 9, or 10 carbon atoms, and including 5-and 6-membered hydrocarbon and heterocyclic aromatic rings.

The aryl group can be optionally substituted (a “substituted aryl”) withone or more aryl group substituents, which can be the same or different,wherein “aryl group substituent” includes alkyl, substituted alkyl,aryl, substituted aryl, aralkyl, hydroxyl, alkoxyl, aryloxyl,aralkyloxyl, carboxyl, acyl, halo, nitro, alkoxycarbonyl,aryloxycarbonyl, aralkoxycarbonyl, acyloxyl, acylamino, aroylamino,carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, arylthio, alkylthio,alkylene, and —NR′R″, wherein R′ and R″ can each be independentlyhydrogen, alkyl, substituted alkyl, aryl, substituted aryl, and aralkyl.

Thus, as used herein, the term “substituted aryl” includes aryl groups,as defined herein, in which one or more atoms or functional groups ofthe aryl group are replaced with another atom or functional group,Including for example, alkyl, substituted alkyl, halogen, aryl,substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino,dialkylamino, sulfate, and mercapto.

Specific examples of aryl groups include, but are not limited to,cyclopentadienyl, phenyl, furan, thiophene, pyrrole, pyran, pyridine,imidazole, benzimidazole, isothiazole, isoxazole, pyrazole, pyrazine,triazine, pyrimidine, quinoline, isoquinoline, indole, carbazole, andthe like.

“Cyclic” and “cycloalkyl” refer to a non-aromatic mono- or multicyclicring system of about 3 to about 10 carbon atoms, e.g., 3, 4, 5, 6, 7, 8,9, or 10 carbon atoms. The cycloalkyl group can be optionally partiallyunsaturated. The cycloalkyl group also can be optionally substitutedwith an alkyl group substituent as defined herein, two, and/or alkylene.There can be optionally inserted along the cyclic alkyl chain one ormore oxygen, sulfur or substituted or unsubstituted nitrogen atoms,wherein the nitrogen substituent is hydrogen, alkyl, substituted alkyl,aryl, or substituted aryl, thus providing a heterocyclic group.Representative monocyclic cycloalkyl rings include cyclopentyl,cyclohexyl, and cycloheptyl. Multicyclic cycloalkyl rings includeadamantyl, octahydronaphthyl, decalin, camphor, camphane, andnoradamantyl.

“Alkoxyl” refers to an alkyl-O— group wherein alkyl is as previouslydescribed. The term “alkoxyl” as used herein can refer to, for example,methoxyl, ethoxyl, propoxyl, isopropoxyl, butoxyl, t-butoxyl, andpentoxyl. The term “oxyalky” can be used interchangeably with “alkoxyl”.

“Aralkyl” refers to an aryl-alkyl-group wherein aryl and alkyl are aspreviously described, and included substituted aryl and substitutedalkyl. Exemplary aralkyl groups include benzyl, phenylethyl, andnaphthylmethyl.

“Alkylene” refers to a straight or branched bivalent aliphatichydrocarbon group having from 1 to about 20 carbon atoms, e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbonatoms. The alkylene group can be straight, branched or cyclic. Thealkylene group also can be optionally unsaturated and/or substitutedwith one or more “alkyl group substituents.” There can be optionallyinserted along the alkylene group one or more oxygen, sulfur orsubstituted or unsubstituted nitrogen atoms (also referred to herein as“alkylaminoalkyl”), wherein the nitrogen substituent is alkyl aspreviously described. Exemplary alkylene groups include methylene(—CH₂—); ethylene (—CH₂—CH₂—); propylene (—(CH₂)₃—); cyclohexylene(—C₆H₁₀—); —CH═CH—CH═CH—; —CH═CH—CH₂—; —(CH₂)_(q)—N(R)—(CH₂)—, whereineach of q and r is independently an integer from 0 to about 20, e.g., 0,1, 2, 3, 4, 5, 6, 7, 5, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or20, and R is hydrogen or lower alkyl; methylenedioxyl (—O—CH₂—O—); andethylenedioxyl (—O—(CH₂)₂—O—). An alkylene group can have about 2 toabout 3 carbon atoms and can further have 6-20 carbons.

“Arylene” refers to a bivalent aryl group. An exemplary arylene isphenylene, which can have ring carbon atoms available for bonding inortho, meta, or para positions with regard to each other, i.e.,

respectively. The arylene group can also be napthylene. The arylenegroup can be optionally substituted (a “substituted arylene”) with oneor more “aryl group substituents” as defined herein, which can be thesame or different.

“Aralkylene” refers to a bivalent group that contains both alkyl andaryl groups. For example, aralkylene groups can have two alkyl groupsand an aryl group (i.e., -alkyl-aryl-alkyl-), one alkyl group and onearyl group (i.e., -alkyl-aryl-) or two aryl groups and one alkyl group(i.e., -aryl-alkyl-aryl-).

The term “amino” and “amine” refer to nitrogen-containing groups such asNR₃, NH₃, NHR₂, and NH₂R, wherein R can be alkyl, branched alkyl,cycloalkyl, aryl, alkylene, arylene, aralkylene. Thus, “amino” as usedherein can refer to a primary amine, a secondary amine, or a tertiaryamine. In some embodiments, one R of an amino group can be adiazeniumdiolate (i.e., NONO).

The terms “cationic amine” and “quaternary amine” refer to an aminogroup having an additional (i.e., a fourth) group, for example ahydrogen or an alkyl group bonded to the nitrogen. Thus, cationic andquarternary amines carry a positive charge.

The term “alkylamine” refers to the -alkyl-NH₂ group.

The term “carbonyl” refers to the —(C═O)— group.

The term “carboxyl” refers to the —COOH group and the term “carboxylate”refers to an anion formed from a carboxyl group, i.e., —COO″.

The terms “halo”, “halide”, or “halogen” as used herein refer to fluoro,chloro, bromo, and iodo groups.

The term “hydroxyl” and “hydroxy” refer to the —OH group.

The term “hydroxyalkyl” refers to an alkyl group substituted with an —OHgroup.

The term “mercapto” or “thio” refers to the —SH group.

The term “silyl” refers to groups comprising silicon atoms (Si).

As used herein the term “alkoxysilane” refers to a compound comprisingone, two, three, or four alkoxy groups bonded to a silicon atom. Forexample, tetraalkoxysilane refers to Si(OR)₄, wherein R is alkyl. Eachalkyl group can be the same or different. An “alkylsilane” refers to analkoxysilane wherein one or more of the alkoxy groups has been replacedwith an alkyl group. Thus, an alkylsilane comprises at least onealkyl-Si bond. The term “fluorinated silane” refers to an alkylsilanewherein one of the alkyl groups is substituted with one or more fluorineatoms. The term “cationic or anionic silane” refers to an alkylsilanewherein one of the alkyl groups is further substituted with an alkylsubstituent that has a positive (i.e., cationic) or a negative (i.e.anionic) charge, or can become charged (i.e., is ionizable) in aparticular environment (i.e., in vivo).

The term “silanol” refers to the Si—OH group.

II. NITRIC OXIDE-RELEASING PARTICLES

The presently disclosed subject matter provides nitric oxide-releasingparticles and their use in biomedical and pharmaceutical applications.In many embodiments, the presently disclosed particles release nitricoxide in a controlled and/or a targeted manner and thereby improve andprolong the biological action and specificity of nitric oxide. In someembodiments, the presently disclosed nitric oxide-releasing particlescan be functionalized to provide a new platform for the delivery ofnitric oxide to cells and/or tissues in vivo. Thus, the presentlydisclosed nitric oxide-releasing particles provide a unique scaffold fornitric oxide donor chemistry and nitric oxide release therapy.

Referring now to FIG. 1, the presently disclosed particles P can, insome embodiments, be described in terms of comprising a core CR, anitric oxide donor NO, an “interior” or “interior region” IR whichcomprises the area inside the exterior, and an “exterior” or an“exterior region” ER. As described more fully hereinbelow, interior IRcan also contain organic linker 0LK that can optionally include a labileportion or group LP.

Exterior or exterior region ER can be defined as the outermost chemicalfunctionality of particle P. In some embodiments, exterior ER contains amoiety or moieties that can control the nitric oxide release kinetics ofparticle P, alter the biocompatibility of particle P, manipulate thesolubility of particle P, provide for the targeted delivery of particleP to a desired location (e.g., a specific cell, tissue or organ) priorto NO-release, provide for imaging or tracking of particle P, or supplyan additional therapeutic agent (i.e., in addition to the NO). Such anexterior ER can be said to control a function of NO-releasing particleP, or be “functionalized.” In some embodiments, the chemical groups ofexterior region ER can control more than one of the functions ofNO-releasing particle P, and exterior ER can be described as“multi-functional.” In some embodiments, chemical moieties or otherstructural characteristics throughout particle P (e.g., in core CR orinterior IR) can be used to control a factor or factors related toNO-release kinetics, particle solubility, targeting, imaging, tracking,additional therapeutic ability, or biocompatibility, and entire particleP can be described as multi-functional.

As shown in FIG. 1, in some embodiments, interior region. IR comprisesorganic linker LK. As used herein, the term “organic linker” or “linker”refers to an organic tether bridging the gap between the particle coreand the particle exterior. In some embodiments, as described more fullyhereinbelow, organic linker LK can comprise labile group LP. In someembodiments, organic linker LK can be somewhat or substantiallyhydrophobic. In some embodiments, linker LK is branched. In someembodiments, linker LK is covalently attached to one or more of theother elements of particle P, such as core CR, exterior ER or NO donorN.

The particles of the presently disclosed subject matter can be anyshape. Thus, the particles can be spherical, elliptical, or amorphous.The size and shape of the particles is, at least in part, determined bythe nature (i.e., the chemical composition) or the method of synthesisof the core. In some embodiments, the size of the particle can bemanipulated to affect the amount or rate of NO-release.

In some embodiments, the NO-releasing particles are nanoparticles. Insome embodiments, the term “nanoparticle” Is meant to refer to aparticle having a diameter of between about 0.5 rim and about 1000 nm.In some embodiments, the nanoparticles have a diameter of between about1 nm and about 500 nm. In some embodiments, the nanoparticles can have adiameter of between about 2 nm and about 200 nm. In some embodiments,the particles have a diameter of between about 1 nm and about 50 nm.

In some embodiments, the particles are larger than 1000 nm. Thus, insome embodiments, the particle is a microparticle. In some embodiments,the particles have a diameter of up to about 25 microns. In someembodiments, the particle can have a diameter of up to about 100microns.

The nitric oxide donor can be part of the core, the interior, or theexterior of the particle. The NO donor can be encapsulated in one of thecore, the interior, or the exterior. The NO donor can be associated witha particular region of the particle via non-covalent interactions suchas Van der Waals interactions, electrostatic interactions (such asinteractions between dipoles or between charged groups), hydrogenbonding, or combinations thereof. Further, the NO donor can becovalently bonded to one of the core, the interior, or the exterior. Thepercent composition of the NO releasing moiety can be varied viacovalent attachment or via encapsulation to impart an effective payloadof nitric oxide for the desired therapeutic or other result.

The NO releasing moiety or NO donor is engineered in such a fashion asnot to disrupt the other particle descriptors while storing its quantityof NO until the appropriate targeting of the particle has occurred. TheNO release can be initiated thermally or via any of the degradationstrategies for the labile portion of the linker as described hereinbelow. Thus the NO donor can be any moiety capable of releasing NO,including N-diazeniumdiolates, nitrosamines, hydroxyl nitrosamines,nitrosothiols, hydroxyl amines, hydroxyureas, metal complexes, organicnitrites and organic nitrates. See, Wang, P. G., et al., Nitric OxideDonors: For Pharmaceutical and Biological Applications; Wiley-VCH:Weinheim, Germany, 2005; and Wang. P. G., et al., Chem. Rev., 102,1091-1134 (2002).

In some embodiments, the NO donor is a N-diazeniumdiolate (i.e., a1-amino-substituted deazen-1-lum-1,2-diolate), N-Diazeniumdiolates areparticularly attractive as NO donors due to their ability to generate NOspontaneously under biological conditions. See Hrabie, J. A. and Keefer,L. K., Chem. Rev., 102, 1135-1154 (2002); and Napoli, C. and lanarro, L.J., Annu. Rev. Pharmacol. Toxicol., 43, 97-123 (2003). As shown inScheme 1, below, several N-diazeniumdiolate compounds have beensynthesized using a range of nucleophilic residues that encompassprimary and secondary amines, polyamines, and secondary amino acids, SeeHrabie, J. A., and Keefer L. K., Chem. Rev., 102, 1135-1154 (2002). Inthe formation of the N-diazeniumdiolate, one equivalent of amine reactswith two equivalents of nitric oxide under elevated pressure. A base(e.g., an alkoxide like methoxide) removes a proton from the aminenitrogen to create the anionic, stabilized [N(O)NO] group. While stableunder ambient conditions, N-diazeniumdiolates decompose spontaneously inaqueous media to generate NO at rates dependent upon pH, temperature,and/or the structure of the amine moiety. For example,N-diazeniumdiolate-modified proline (PROLI/NO),2-(dimethylamino)-ethylputreamine (DMAEP/NO), N,N-dimethylhexanediamine(DMHD/NO), and diethylenetriamine (DETA/NO) have been developed as smallmolecule NO donors with diverse NO release half-lives ranging from 2seconds to 20 hours at pH 7.4 and 37° C. See Hrabie, J. A., and Keefer,L. K., Chem. Rev., 102, 1135-1154 (2002); and Keefer, L. K., Annu, Rev.Pharmacol. Toxicol 43, 585-607 (2003).

As described in more detail immediately hereinbelow, in someembodiments, the “core” of the presently disclosed particles comprises amaterial selected from the group including, but not limited to: (a) ametallic cluster; (b) a dendritic network; and (c) a co-condensed silica(i.e. siloxane-bonded) network possessing variable silane functionality.

II.A. Cores Comprising Metallic Clusters

In some embodiments, the core of the presently disclosed particlescomprises a metallic cluster. The metallic clusters can comprise anymetallic complex that can be passivated or “protected” for furtherfunctionalization. For example, protected metallic complexes can beformed, in some embodiments, by being coated with organic polymers orsilica. Metallic complexes can also be protected with monolayers oforganic molecules wherein the organic molecules contain a functionalitythat coordinates to or otherwise forms a covalent or non-covalent bondwith metal atoms at the surface of the metallic complex.

The metallic complexes can be metals, metal alloys, metal salts, ormetal oxides. In some embodiments, the metallic complex comprises gold,sliver, platinum, iron oxide (i.e., FeO, Fe₂O₃, or Fe₃O₄), orsemiconductor particles such as CdSe, and the like. In some embodimentsthe iron oxide is magnetite (i.e., Fe₃O₄). In some embodiments, the coreIs a monolayer protected gold cluster, which can be formed via a varietyof methods known in the art, including the Brust method and theSchufz-Dobrick method.

Monolayer protected cluster (MPC) gold nanoparticles or MPCs, see Brust,M., J. Chem. Soc., Chem: Comm., 801-602 (1994), have received muchattention due to their unique size (1 nm to 5 nm), stability, and highlyfunctional design. See Feldheim, D. L. and Foss, C. A., eds, MetalNanoparticles—Synthesis Characterization, and Applications, MarcelDekker, Inc: New York, p. 360 (2000). As shown in FIG. 2, the exteriorof MPCs can be altered by place exchanging in other thiols containingdesired functional groups. See Hostetler, M. I., et al., Langmuir, 15,3782-3789 (1999).

Further functionalization of the particles with receptor molecules toenable specific antibody-antigen or ligand-receptor interactions allowsfor the targeting of specific tissues or cells. The size and stabilityof NO-releasing MPC gold nanoparticles provides for a range ofbiomedical and pharmaceutical applications including in vivo sensordesign and topical creams to enhance wound healing and/or dilate bloodvessels below the skin.

III.B. Cores Comprising Dendrimers

Dendrimers provide a unique scaffold for nitric oxide donor chemistrywhereby the multivalent dendritic exterior can be functionalized to suitany number of materials science or biomedical applications.

Dendrimers are polymers with densely branched structures having a largenumber of reactive groups. A dendritic polymer includes several layersor generations of repeating units which all contain one or more branchpoints. Dendrimers, including hyperbranched dendritic polymers, areprepared by condensation reactions of monomeric units having at leasttwo reactive groups. Dendrimers generally consist of terminal surfacegroups, interior branch junctures having branching functionalitiesgreater than or equal to two, and divalent connectors that covalentlyconnect neighboring branching junctures.

Dendrimers can be prepared by convergent or divergent synthesis.Divergent synthesis of dendrimers involves a molecular growth processthat occurs through a consecutive series of geometrically progressivestep-wise additions of branches upon branches in a radially outwarddirection to produce an ordered arrangement. Thus, each dendriticmacromolecule can be said to include a core cell, one or more layers ofinternal cells, and an outer layer of surface cells, wherein each of thecells includes a single branch juncture. The cells can be the same ordifferent in chemical structure and branching functionality. The surfacebranch cells may contain either chemically reactive or passivefunctional groups, Chemically reactive surface groups can be used forfurther extension of dendritic growth or for modification of dendriticmolecular surfaces. The chemically passive groups may be used tophysically modified dendritic surfaces, such as to adjust the ratio ofhydrophobic to hydrophilic terminals, and/or to improve the solubilityof the dendritic polymer for a particular solvent.

The convergent synthesis of dendrimers involves a growth process thatbegins from what will become the surface of the dendron or dendrimer andprogresses radially toward a focal point or core. The dendritic polymersmay be ideal or non-ideal, i.e., imperfect or defective. Imperfectionsare normally a consequence of either incomplete chemical reactions, orunavoidable competing side reactions. In practice, real dendriticpolymers are generally non-ideal, i.e., contain certain amounts ofstructural imperfections.

Hyperbranched dendritic networks refer to a class of dendritic polymersthat contain high levels of non-ideal irregular branching. Specifically,hyperbranched polymers contain a relatively high number of irregularbranching areas in which not every repeat unit contains a branchjuncture. The preparation and characterization of dendrimers, dendrons,random hyperbranched polymers, controlled hyperbranched polymers, anddendrigrafts is well known. Examples of dendimers and dendrons, andmethods of synthesizing the same are set forth in U.S. Pat. Nos.4,507,466; 4,558,120; 4,568,737; 4,587,329; 4,631,337; 4,694,064;4,713,975; 4,737,550; 4,871,779 and 4,857,599. Examples of hyperbranchedpolymers and methods of preparing the same are set forth, for example inU.S. Pat. No. 5,418,301.

Suitable dendrimers for use as core scaffolds of the presently disclosedparticles include polypropylenimine dendrimer; polyamidoamine (PAMAM)dendrimer; polyaryl ether dendrimer; polylysine dendrimer; polyesterdendrimer; polyamide dendrimer; dendritic polyglycerol; and triazinedendrimers,

In some embodiments, the presently disclosed subject matter provides aseries of polypropylenimine (PPI) dendrimer conjugates, which compriseexterior secondary amines. The secondary amine-containing PPI dendrimerscan be synthesized from PPI dendrimers having exterior primary amines byacylating the primary amines and reducing the carbonyl of the resultingamide groups to form secondary amines. Alternatively, the primary aminescan be acylated with groups already containing a secondary amine. Farexample, the exterior primary amines of a PPI dendrimer can be acylatedwith praline.

The secondary amine functional group of the dendrimers is converted inhigh yields to a nitric oxide donor in the presence of a strong base andgaseous nitric oxide. As provided herein, the dendrimer size and surfacefunctionality effect both the percent conversion of the secondary amineto the nitric oxide donor and the nitric oxide release kinetics.

II.C. Cores Comprising Co-Condensed Silica Networks

Inorganic-organic hybrid silica nanoparticles, functionalized ceramiccomposites prepared from silicon dioxide, have been explored forapplications spanning separation, biological labeling, diagnostics, andcarrier systems for the controlled delivery of drugs, genes, andproteins. See Lai, C.-Y., et al., J. Am. Chem. Soc., 125, 4451-4459(2003); Munoz. B., et al., Chem. Mater., 15, 500-503 (2003); Roy, I., etal., Proc. Natl. Acad. Sci, U.S.A., 102, 279-284 (2005); Trewyn, B. G.,et al., Nano. Lett., 4, 2139-2143 (2004); and Yoshitake, H., New. J.Chem., 29, 1107-1117 (2005). The drug delivery potential of silicaparticles has received much attention because of their physical andchemical versatility and non-toxic nature. See Sayari, A., and Hambudi,S., Chem. Mater., 13, 3151-3168 (2001); and Stein, A., et al., Adv.Mater., 12, 1403-1419 (2000). The synthesis of inorganic-organic hybridsilica modified with reactive organic groups (e.g., amines,carboxylates, thiols, olefins, halides, and epoxides) capable of furtherfunctionalization with deliverable molecules has been reported, SeeSavari, A., and Hamoudi, S., Chem. Mater., 13, 3151-3168 (2001); andStein, A., et al., Adv. Mater., 12, 1403-1419 (2000), Indeed, numeroussilane-coupling agents with the aforementioned functional moieties havebeen developed for surface grafting (via free silanol groups) of drugsand other therapeutics. See Anwander, R., et al., Stud. Surf Sci.Catal., 117, 135-142 (1998).

In one example, Meyerhoff and coworkers have reported graftingamine-functionalized silylation reagents onto the surface of fumedsilica (amorphous particles, 0.2-0.3 gm in diameter). See Zhane, H., etal., J. Am. Chem. Soc., 125, 5015-5024 (2003). The surface bound amineswere then converted to N-diazeniumdiolate NO donors. The NO-releasingsilica was employed as filler for preparing silicone rubber polymercoatings with improved hemocompatibility.

The usefulness of such scaffolds as therapeutic NO delivery systemsremains hindered for multiple reasons. Since the modification isrestricted to the outer surface of the particles, the NO storagecapability is inherently limited, control over the NO release kineticsis problematic, and NO donor moieties are more susceptible tocontamination from reactive species (e.g., radicals, peroxides, andtransition metals) in biological fluids. See Keefer, L. K., Anna, Rev.Pharmacot. Toxicol., 43, 585-607 (2003); Naooli. C., and lonarro, L. J.,Annu. Rev. Pharmacol. Toxicol., 43, 97-123 (2003); and Zhou, Z., andMeyerhoff, M. E., Biomacromolecules, 6, 780-789 (2005).

The particles of the presently described subject matter can compriseco-condensed silica networks that provide NO-delivery systems ofincreased NO storage capacity and an enhanced ability to control NOrelease kinetics. In some embodiments, the presently disclosedNO-releasing silica-based particles are prepared via a “one-pot”synthetic strategy. See Stein, A., et al., Adv. Mater. 12, 1403-1419(2000); Hatton, B. et al., Acc. Chem. Res., 38, 305-312 (2005), Lin,H.-P., and Mou, C,-Y., Acc. Chem. Res., 35, 927-935 (2002). Thus, asshown in FIG. 3, the inorganic-organic hybrid silica particles areprepared via a sol-gel process involving the co-condensation oftetraethyl orthosilicate (TEOS) or another alkoxysilane with di- ortri-aminoalkoxysilaries. The “sol-gel” process involves two types ofchemical reactions; a hydrolysis reaction in which an alkoxy group of analkoxysilane is hydrolyzed to a silanol (i.e., a hydroxy group attachedto the Si atom), followed by a condensation reaction wherein twosilanols or a silanol and an alkoxysilane react to form a siloxane bond(i.e., Si—O—Si).

The advantage of a “one-pot” approach is that the N-diazeniumdiolate NOdonor precursors (i.e., the amino groups of the di- andtri-aminoalkoxysilane) can be distributed uniformly throughout theentire particle as opposed to only at the surface as is the case foramine-modified silica particles formed via surface grafting methods. SeeFIGS. 4A and 4B. Indeed, the direct “one-pot” synthesis provides betterstructural stability and more straightforward control over the amount oforganoalkoxysilanes incorporated in the silica structure: See Stein, A.,et al., Adv. Mater., 12, 1403-1419 (2000); and Lim, M, H., and Stein,A., Chem, Mater., 11, 3285-3295 (1999). Further, additional silanescontaining a variety of other functional groups can also be co-condensedinto the structure, thereby affecting the size, the solubility, or theporosity of the particles.

Thus, in some embodiments, the nanoparticle core comprises aco-condensed silane network formed from the co-condensation of analkoxysilane and an aminoalkoxysilane. In some embodiments, theaminoalkoxysilane is further functionalized after the co-condensation bytreatment with nitric oxide so that the amines are transformed intoN-diazeniumdiolates. See FIG. 5A. In some embodiments, theaminoalkoxysilane is “pretreated” or “precharged” with nitric oxideprior to co-condensation with the alkoxysilane. See FIG. 5B. The“pre-charging” method can be used to create a co-condensed silicaparticle more densely functionalized with NO-donors.

In some embodiments, the alkoxysilane is a tetraalkoxysilane having theformula Si(OR)₄, wherein R is an alkyl group. The R groups can be thesame or different. In some embodiments the tetraalkoxysilane is selectedtetra methyl orthosilicate (TMOS) or tetraethyl orthosilicate (TEOS).

In some embodiments, the aminoalkoxysilane has the formula:R″—(NHR′)_(n)—Si(OR)₃wherein R is alkyl, R′ is alkylene, branched alkylene, or aralkylene, nis 1 or 2, and R″ is selected from the group consisting of alkyl,cycloalkyl, aryl, and alkylamine. In some embodiments, theaminoalkoxysilane can be selected fromN-(6-aminohexyl)aminopropyltrimethoxysilane (AHAP3);N-(6-aminoethyl)aminopopyltrimethoxysilane;(3-trimethoxysilylpropyl)diethylenetriamine (DET3);(aminoethylaminomethyl)phenethyltrimethoxysilane (AEMP3);[3-(methylamino)propyltrimethoxysilane;N-butylamino-propyltrimethoxysilane;N-ethylaminoisobutyltrimethoxysilane;N-phenylamino-propyltrimethoxysilane; andN-cyclohexylaminopropyltrimethoxysilane. The structures ofrepresentative suitable aminoalkoxysilanes are shown in FIG. 6.

In some embodiments, the aminoalkoxysilane has the formula:NH[R′—Si(OR)₃]₂,wherein R is alkyl and R′ is alkylene. Thus, in some embodiments theaminoalkoxysilane can be selected frombis-[3-(trimethoxysilyl)propyl]amine andbis-[(3-trimethoxysifyl)propyl]ethylenediamine.

In some embodiments, as described hereinabove, the aminoalkoxysilane isprecharged for NO-release and the amino group is substituted by adiazeniumdiolate. Therefore, in some embodiments, the aminoalkoxysilanehas the formula:R—N(NONO⁻X⁴)—R′—Si(OR)₃,wherein R is alkyl, R′ is alkylene or aralkylene, R″ is alkyl oralkylamine, and X⁺ is a cation selected from the group consisting ofNa⁺, K⁺, and Li⁺.

The composition of the silica network, (e.g., amount or the chemicalcomposition of the aminoalkoxysilane) and the nitric oxide chargingconditions (e.g., the solvent and base) can be varied to optimize theamount and duration of nitric oxide release. Thus, in some embodiments,the composition of the presently disclosed silica particles can bemodified to regulate the half-life of NO release from silica particles.

In some embodiments, the hydrophobicity of nitric oxide-releasing silicaparticles can be controlled by co-condensing silane precursors having avariety of functional groups into the co-condensed silica network. Insome embodiments, the other silane precursors are selected from thegroup including but not limited to alkylsilanes, positively chargedsilanes, negatively charged silanes, and fluorinated silanes. in someembodiments the other silane precursors can be selected from(heptadecafluoro-1,1,2,2-tetrahydrodecyl)triethoxysilane;(3,3,3-trifluoro-propyl)trimethoxysilane;(perfluoroalkyl)ethyltriethoxysilane;N—N-didecyl-N-methyl-N-(3-trimethoxysilyl)ammonium chloride;octadecyldimethyl-(3-trimethoxysilylpropyl)ammonium chloride;3-trihydroxysllylpropylmethyl phosphonate, sodium salt;carboxylethylsilanetriol, sodium salt; methyltrimethoxysilane;butyltrimethoxysilane; butyltriethoxysilane; propyltrimethoxysilane; andoctadecyltrimethoxysilane.

In some embodiments, the co-condensed silica network comprises (i.e., isformed from the condensation of a solution containing) between about 10mol % to about 99 mol % of tetraalkoxysliane; about 1 mol % to about 90mol % of aminoalkoxysilane; about 0 mol % to about 20 mol % offluorinated silane; about 0 mol % to about 20 mol % of cationic oranionic silane; and about 0 mol % to about 20 mol % of alkylsilane.

In some embodiments, the porosity and the NO-release capability of thesilica network can be controlled by co-condensing silanes in thepresence of a templating component. Such templating components caninclude surfactants and micelles. After condensation of the silicanetwork, the templating component can be removed, leaving pores in thesilica. The incorporation of pores in a NO-releasing silica particle canincrease the surface area available for NO donor loading or can serve toincrease the rate of NO release by increasing the accessibility of waterto the NO donors.

For example, FIG. 7 shows the schematic representation of the synthesisof a mesoporous silica network using micelles as pore templates. Asshown in FIG. 7, micelles can self-associate in a controlled solventenvironment to form an ordered three-dimensional structure, such as amicellular rod, or an even more highly structured array of multiplerods. Solutions containing mixtures of silanes can be introduced intothe micelle solution and condensed, surrounding, but not penetrating,the micelle rods. Following condensation of the silane mixture, themicelles can be removed from the condensed silica via solventextraction, leaving behind pores in the silica.

In some embodiments, the presently disclosed subject matter providesfunctionalized silicas, silicas that can be further elaborated through avariety of chemical coupling reactions known in the art. In someembodiments, the functionalized silica is an amino-modified silica. Insome embodiments, the functionalized silica is an epoxy-modified silica.

In some embodiments, the presently disclosed silica chemistry iscombined with hydroxylamine chemistry. In some embodiments, thepresently disclosed silica chemistry is combined with hydroxyureachemistry.

III. TRIGGERED RELEASE OF NITRIC OXIDE FROM NITRIC OXIDE-RELEASINGPARTICLES

Controlled and/or targeted delivery techniques typically enhance theefficacy and/or safety of an active agent by controlling the rate and/orlocation of the release of the active agent. In some embodiments, therelease of nitric oxide from the presently disclosed nitricoxide-releasing particles can be selectively turned on or turned off(i.e., triggered), as desired.

In some embodiments, the organic linker comprises a “labile” portion. Insuch embodiments, the triggered degradation of the linker can affect themechanism, quantity, rate, and duration of NO release. Referring toFIGS. 1 and 8, labile portion LP of linker LK can be placed at variablepositions A, B, or C, in relation to exterior ER such that the positionof linker LK further affects the mechanism, quantity, rate, and durationof NO release. For example, in some embodiments, position A of FIG. 8can be adjacent to NO donor NO in interior IR of NO-releasing particle Pof FIG. 1; position B can be centrally located between NO donor NO andexterior ER; and position C can be located in close proximity toexterior ER. Thus, in some embodiments, a labile group LP at position Ccan be degraded more quickly by environmental conditions to whichparticle P is subjected, in turn exposing NO donor NO located ininterior IR of particle P to the same environmental conditions sooner.Labile groups LP located more deeply in particle interior IR atpositions A or B can, in some embodiments, provide for prolonged ordelayed release kinetics.

In some embodiments, the “labile” portion of the linker can be degradedby exposure to a stimulus, e.g., via a triggering mechanism. In someembodiments, the stimulus, or triggering mechanism, is selected from thegroup including but not limited to pH, light, and enzymatic action.

In embodiments wherein decomposition of the labile portion of the linkeris triggered by pH, the linker comprises functionalities, such asesters, hydrazones, acetals, and/or other organic functional groups,which are responsive to changes in pH. Accordingly, in some embodiments,the linker decomposes in a predetermined pH range. More particularly, insome embodiments, the linkers are designed to utilize the pH ofincreased acidity inside an endosome, the cellular structure resultingfrom internalization of a macromolecule via endocytosis.

In some embodiments, decomposition of the linker is triggered byexposure to light. In such embodiments, the “labile” portion is subjectto photocleavage, such that a photolabile moiety is built into thevariable linker that results in degradation of the linker structure uponexposure to light.

In some embodiments, an enzyme substrate is incorporated into the linkerto impart specificity of the system to a desired enzyme environment ofinterest, followed by degradation of the linker via the enzymaticpathway of interest.

Thus, in some embodiments, the lability of the linker can be used as astrategy to control the mechanism, quantity, rate, and duration of NOrelease from the NO-releasing moiety. Labile linkers include esters,hydrozones, acetals, thiopropionates, photolabile moieties and aminoacid sequences subject to enzyme degradation.

In some embodiments, the organic linker is a hydrophobic linker. Ahydrophobic linker can be chosen as an approach for protecting the NOdonor, for example the diazeniumdiolate, and/or the labile linker fromcontact with water or protons when the particle is placed in an aqueousenvironment. The length and exact chemical composition of a hydrophobiclinker can, therefore, be used to control the NO-release kinetics. Theterm hydrophobic can include groups that are strongly hydrophobic (i.e.,have a very low dielectric constant) or are only somewhat hydrophobic(i.e., would allow water to slowly penetrate into the interior of theparticle).

Alternatively, the organic linker can be amphiphilic, containing bothhydrophobic and hydrophilic groups. Such a linker might provide channelsin the interior of the particle, thereby enhancing solvent access to alabile linker or a NO-donor.

NO release can also be controlled through encapsulation of the NO-donorin a carrier system, such as a nano- or microparticle, a cell, a cellghost, a lipoprotein, a liposome, a micelle, a microbubble, amicrosphere, or a particle made at least partially of insoluble orbiodegradable natural or synthetic polymers. In such a system, the NOcan be gradually released as the carrier degrades in the body. The rateof degradation typically varies responsively to conditions in thesubject, such as temperature, pH level, and enzymatic activity. Thus,through the use of such delivery techniques, a sustained release of thetherapeutic agent can be maintained for long periods of time.

IV. ADDITIONAL FUNCTIONALIZATION OF THE NITRIC OXIDE RELEASING PARTICLES

As provided herein, the exterior, interior and/or core of the presentlydisclosed particles can be functionalized to impart biocompatibility,alter pharmacokinetic behavior, convey targeting functionality, addadditional therapeutic components, and impart imaging capability,relevant to the delivery and study of the NO as a therapeutic. In someembodiments, the exterior of the particle can be functionalized with oneor more chemical or biomolecular moieties.

The exterior can be of uniform or variable chemical composition. In someembodiments, the functionalization of the exterior of the particle cancomprise the addition of a layer or coating surrounding the interior ofthe particle. In some embodiments, the functionalization can involve theaddition of one or more pendant groups to individual points on theperiphery of the particle. Thus, the exterior can comprise one or morependant antigens for particle targeting as discussed more fully hereinbelow. The exterior can also comprise individual chemical moieties thataffect solubility, such as hydroxy groups, thiols, methyl-terminatedalkyl chains, sulfonates, phosphates, carboxylates, and cationic orquaternary amines. Further, the exterior can comprise a polymeric layer,for example a hydrophilic polymer to impart improved aqueous solubilityor a known biocompatible polymer. The polymeric layer can be abiodegradable polymer, which can protect the NO donor from water for aperiod of time when used either in vivo or in vitro. Such a polymercoating can thereby affect the NO-release kinetics by allowing forcontinued NO-release over time as the polymer coating degrades. Suitablepolymers for functionalizing the exterior of the presently describedparticles include (poly)ethyleneoxide, (poly)urethanes,N-(2-hydroxypropyl) methacrylamide copolymers, and lactide/glycolidecopolymers (e.g. PLGA).

IV.A. Nitric Oxide Releasing Particles for Targeted Delivery of NitricOxide

In some embodiments, additional functionalization of the particleenables targeting of specific cells, tissues, or organs. Thus, in someembodiments, the presently disclosed nitric oxide-releasing particlescan be further modified by attaching selective recognition agents to thesurface or exterior thereof. Such selective recognition agents include,but are not limited to small molecule ligands; biomolecules, such asantibodies and antibody fragments; and other agents such as cytokines,hormones, carbohydrates, sugars, vitamins, and peptides.

A specific targeting moiety is not required in all cases. In someembodiments, the site specific targeting can also include a more passiveapproach, such as the enhanced permeability and retention effect (EPR)associated with tumor vasculature. Site specific targeting can also beaccomplished by the used of NO-release particles containing linkers thattrigger release of the nitric oxide only upon contact with enzymesspecific to a disease state or to a particular organ or tissue. Finally,targeting can be accomplished via localized delivery of the particles,for example, topically directly to a wound, or through injectiondirectly to a tumor site.

Generally, when a particle targets cells through a cell surface moietyit is taken into the cell through receptor-mediated endocytosis. Anymoiety known to be located on the surface of target cells (e.g. tumorcells) finds use with the presently disclosed particles. For example, anantibody directed against such a cell surface moiety can be used.Alternatively, the targeting moiety can be a ligand directed to areceptor present on the cell surface or vice versa.

In particle embodiments using a specific targeting moiety (i.e., aparticle-associated moiety designed to direct the particle to a specificcell, tissue or organ), the targeting moiety is optionally associatedwith the exterior of the particle. The targeting moiety can beconjugated, directly to the exterior via any useful reactive group onthe exterior, such as, for example, an amine, an alcohol, a carboxylate,an isocyanate, a phosphate, a thiol, a halide, or an epoxide. Forexample, a targeting moiety containing or derivatized to contain anamine that is not necessary for the recognition of the moeity with thetargeted cell can be coupled directly to a carboxylate present on theparticle exterior using carbodimide chemistry. The targeting moiety canalso be linked to a reactive group on the exterior of the particlethrough a short bi-functional linker, such asN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP, commerciallyavailable from Pierce Chemical Company, Rockford, Ill., United States ofAmerica). Alternatively, a longer bifunctional linker can be used, sucha polyethylene glycol (PEG)-based bifunctional linker commerciallyavailable from EMD Biosciences, Inc. (La Jolla, Calif., United States ofAmerica) or Shearwater Polymers (Huntsville, Ala., United States ofAmerica).

Targeting moieties for use in targeting cancer cells can be designedaround tumor specific antigens including, but not limited to,carcinoembryonic antigen, prostate specific antigen, tyrosinase, ras, asialyly lewis antigen, erb, MACE-1, MAGE-3, BAGE, MN, gp100, gp75, p97,proteinase 3, a mucin, CD81, CID9, CD63; CD53; CD38, CO-029, CA125, GD2,GM2 and O-acetyl GD3, M-TAA, M-fetal or M-urinary find use with thepresently disclosed subject matter. Alternatively the targeting moietycan be designed around a tumor suppressor, a cytosine, a chemokine, atumor specific receptor ligand, a receptor, an inducer of apoptosis, ora differentiating agent. Further, given the importance of theangiogenisis process to the growth of tumors, in some embodiments, thetargeting moiety can be developed to target a factor associated withangiogenisis. Thus, the targeting moiety can be designed to interactwith known angiogenisis factors such as vascular endothelial growthfactor (VEGF). See Brannon-Peppas, L. and Blanchette, J. O., AdvancedDrug Delivery Reviews, 56, 1649-1659 (2004).

Tumor suppressor proteins provided for targeting include, but are notlimited to, p16, p21, p27, p53, p73, Rb, Wilms tumor (WT-1), DCC,neurofibromatosis type 1 (NF-1), von Hippel-Lindau (VHL) disease tumorsuppressor, Maspin, Brush-1, BRCA-1, BRCA-2, the multiple tumorsuppressor (MTS), gp95/p97 antigen of human melanoma, renal cellcarcinoma-associated G250 antigen, KS 1/4 pan-carcinoma antigen, ovariancarcinoma antigen (CA125), prostate specific antigen, melanoma antigengp75, CD9, CD63, CD53, CD37, R2, CD81, CO029, TI-1, L6 and SAS. Ofcourse these are merely exemplary tumor suppressors and it is envisionedthat the presently disclosed subject matter can be used in conjunctionwith any other agent that is or becomes known to those of skill in theart as a tumor suppressor.

In some embodiments, targeting is directed to factors expressed by anoncogene. These include, but are not limited to tyrosine kinases, bothmembrane-associated and cytoplasmic forms, such as members of the Srcfamily, serine/threonine kinases, such as Mos, growth factor andreceptors, such as platelet derived growth factor (PDDG), SMALL GTPases(G proteins) including the ras family, cyclin-dependent protein kinases(cdk), members of the myc family members including c-myc, N-myc, andL-myc and bcl-2 and family members,

Cytokines that can be targeted by the presently disclosed particlesinclude, but are not limited to, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, ILA 1, IL-12, IL-13, IL-14, IL-15, TNF, GM-CSF,8-interferon and y-interferon. Chemokines that can be used include, butare not limited to, M1P1a, M1P1b, and RANTES.

Enzymes that can be targeted include, but are not limited to, cytosinedeaminase, hypoxanthine-guanine phosphoribosyltransferase,galactose-1-phosphate uridyltransferase, phenylalanine hydroxylase,glucocerebrosidase, sphingomyelinase, a-L-iduronidase,glucose-6-phosphate dehydrogenase, HSV thymidine kinase, and humanthymidine kinase.

Receptors and their related ligands that find use in the context of thepresently disclosed subject matter include, but are not limited to, thefolate receptor, adrenergic receptor, growth hormone receptor,luteinizing hormone receptor, estrogen receptor, epidermal growth factorreceptor, fibroblast growth factor receptor, and the like. In someembodiments, the targeting moiety is selected from the group consistingof folic acid, guanidine, transferrin, carbohydrates and sugars. In someembodiments, the targeting moiety is a peptide selected from the groupconsisting of the amino acid sequence RGD and TAT peptides.

For example, folic acid can be a particularly useful targeting moiety intargeting cancer cells. Cancerous tumor cells have an over-expression offolate receptors on their cellular surface. Folic acid (FA) can becovalently bound to the nanoparticle exterior, with varying percentmodification, to impart the FA targeted delivery of the NO releasingnanoparticles. Because of its small size, many folic acid ligands can beattached to the surface of a particle. Wiener has reported thatdendrimers with attached folic acid specifically accumulate on thesurface and within tumor cells expressing the high-affinity folatereceptor (hFR) while control cells lacking hFR showed no significantaccumulation of the folate-derivatized dendrimers. See Wiener, E. C. etal., Invest. Radiol., 32 (12), 748-754 (1997). Folic acid can beattached to amines on the exterior of a particle via a carbodiimidecoupling reaction.

A larger, yet still relatively small targeting moiety is epidermalgrowth factor (EGF), a single-chain peptide with 53 amino acid residues.It has been shown that PAMAM dendrimers conjugated to EGF with thelinker SPDP bind to the cell surface of human glioma cells and areendocytosed, accumulating in lysosomes. See CapAla, J., et al.,Bioconjugate Chem., 7(1), 7-15 (1996). Since EGF receptor density is upto 100 times greater on brain tumor cells compared to normal cells, EGFprovides a useful targeting agent for these kinds of tumors. Since theEGF receptor is also overexpressed in breast and colon cancer, EGF canbe used as a targeting agent for these cells as well. Similarly, thefibroblast growth factor receptors (FGFR) also bind the relatively smallpolypeptides (FGF), and many are known to be expressed at high levels inbreast tumor cell lines (particularly FGF1, 2 and 4). SeePenault-Llorca, F., et al., Int. J. Cancer, 61(2), 170-176 (1995).

Hormones and their receptors include, but are not limited to, growthhormone, prolactin, placental lactogen, luteinizing hormone,follicle-stimulating hormone, chorionic gonadotropin,thyroid-stimulating hormone, leptin, adrenocorticotropin (ACTH),angiotensin I, angiotensin II, β-endorphin, β-melanocyte stimulatinghormone (β-MSH), cholecystokinin, endothelin I, galanin, gastricinhibitory peptide (GIP), glucagon, insulin, amylin, lipotropins, GLP-1(7-37) neurophysins, and somatostatin.

The presently disclosed subject matter contemplates that vitamins (bothfat soluble and non-fat soluble vitamins) placed in the targetingcomponent of the nanodevice can be used to target cells that havereceptors for, or otherwise take up these vitamins. Particularlypreferred for this aspect are the fat soluble vitamins, such as vitaminD and its analogues, vitamin E, Vitamin A, and the like or water solublevitamins such as Vitamin C, and the like.

Antibodies can be generated to allow for the targeting of antigens orimmunogens (e.g., tumor, tissue or pathogen specific antigens) onvarious biological targets (e.g., pathogens, tumor cells, normaltissue). In some embodiments of the presently disclosed subject matter,the targeting moiety is an antibody or an antigen binding fragment of anantibody (e.g., Fab units). Thus, “antibodies” include, but are notlimited to polyclonal antibodies, monoclonal antibodies, chimericantibodies, single chain antibodies, Fab fragments, and an Fabexpression library.

One example of a well-studied antigen found on the surface of manycancers (including breast HER2 tumors) is glycoprotein p185, which isexclusively expressed in malignant cells. See Press, M. F., et al.,Oncogene 5(7), 953-962 (1990). Recombinant humanized anti-HER2monoclonal antibodies (rhuMabHER2) are commercially available under thename HERCEPTIN® from Genentech (South San Francisco, Calif., UnitedStates of America). Other representative antibodies suitable for usewith the presently disclosed subject matter include, but are not limitedto, 1gC-type antibodies, 60bca and J591, which bind to CD14 and prostatespecific membrane antigen (PSMA), see Baker, J. R., Jr.,Biomacrormolecules, 5, 2269-2274 (2004), which is incorporated herein byreference in its entirety, and antibodies F5 and C1, which bind to ErbB2growth factor of breast tumor cell line SK-BR-3.

As described hereinabove, the ability of a particle to provide targeteddelivery of NO is not limited to embodiments involving pendant targetingagents attached to the particle exterior. Non exterior-associatedcharacteristics of the particle also can be used for targeting. Thus, insome embodiments, the enhanced permeability and retention (EPR) effectis used in targeting. The EPR effect is the selective concentration ofmacromolecules and small particles in the tumor microenvironment, causedby the hyperpermeable vasculature and poor lymphatic drainage of tumors.To enhance EPR, in some embodiments, the exterior of the particle can becoated with or conjugated to a hydrophilic polymer to enhance thecirculation half-life of the particle and to discourage the attachmentof plasma proteins to the particle.

In some embodiments, the targeting moiety can be a magnetic moiety, suchas magnetite. In some embodiments, the core of the particle comprisesmagnetite. In some embodiments, the magnetite core is further coatedwith a shell containing a co-condensed silica network that contains orcan be functionalized to contain an NO donor. Once administered to asubject, magnetic particles can be directed to their target, i.e., thesite of desired NO-release, through the application of a magnet. Such amagnet can be applied externally (i.e., outside of the patient orsubject).

For additional exemplary strategies for targeted drug delivery, inparticular, targeted systems for cancer therapy, see Brannon-Peppas, L.and Blanchette, J. O., Advanced Drug Delivery Reviews, 56, 1649-1659(2004) and U.S. Pat. No. 6,471,968, each of which is incorporated hereinby reference in its entirety.

IV.B. Imaging of Nitric Oxide Releasing Particles

In some embodiments, the NO-releasing particle can comprise a moiety toaid in the imaging or tracking of the particles either in viva or exvivo. Tracking of the particles can be useful in determining theefficacy of the nitric oxide release in treating a disease or inassessing the specificity of the targeting of the particle. An imagingor tracking moiety can be associated with any of the core, the interioror the exterior of the particle. In some embodiments, the imaging ortracking moiety is covalently attached to one of the core, the interioror the exterior of the particle. In some embodiments, the tracking agentor moiety is part of the core, for example in particles containingquantum dot cores.

In some embodiments, the tracking of imaging agent is one of afluorescent molecule, an organic dye, or a radioisotope.

In some embodiments, the imaging agent can be a magnetic resonanceimaging (MRI) contrast agent. Thus, in some embodiments, the exterior ofthe particle will be functionalized to contain a group capable ofchelating to a paramagentic ion, for examplediethylenetriaminepentaacetic acid (DTPA), the chelating group of thecommonly used MRI agent Gd(III)-diethylenetriaminepentaacetic acid(Gd(III)-DTPA). Other paramagnetic ions that can be useful in thiscontext of the include, but are not limited to, gadolinium, manganese,copper, chromium, iron, cobalt, erbium, nickel, europium, technetium,indium, samarium, dysprosium, ruthenium, ytterbium, yttrium, and holmiumions and combinations thereof.

IV.C. Additional Therapeutic Agents

In some embodiments, one or more additional therapeutic agents can beused in combination with the NO donor of the presently describedparticles. Such additional agents can be incorporated into the particlesthemselves or be part of a formulation comprising the particles or dosesas a separate formulation prior to, after, or at the same time as aformulation including the particles. Such additional therapeutic agentsinclude, in particular, anti-cancer therapeutics, anti-microbial agents,pain relievers, anti-inflammatories, vasodialators, andimmune-suppressants, as well as any other known therapeutic agent thatcould enhance the alleviation of the disease or condition being treated.

In embodiments wherein the additional therapeutic agent or agents areincorporated into the NO-releasing particles, the additional therapeuticcan be associated with any of the exterior, the interior or the core ofthe particle. For example, the additional agents can be encapsulatedinto the core or linkers in the interior portion of the particle. Theadditional agents can also be covalently attached to the core, theinterior or the exterior of the particles. Further, attachment of theadditional agent can include a triggered release strategy, wherein theadditional agents can be tethered to the particle via a labile linkerthat releases the agent upon contact with water, an increase in pH, orenzymatic or photolytic cleavage, preferably at the desired site ofaction (e.g., a tumor cell, etc.).

The choice of additional therapeutic agents to be used in combinationwith an NO-releasing particle will depend on various factors including,but not limited to, the type of disease, the age, and the general healthof the subject, the aggressiveness of disease progression, and theability of the subject to tolerate the agents that comprise thecombination.

A variety of chemical compounds, also described as “antineoplastic”agents or “chemotherapeutic agents” can be used in combination with orincorporated into the presently disclosed NO-releasing particles used inthe treatment of cancer. Such chemotherapeutic compounds include, butare not limited to, alkylating agents, DNA intercalators, proteinsynthesis inhibitors, inhibitors of DNA or RNA synthesis, DNA baseanalogs, topoisomerase inhibitors, anti-angiogenesis agents, andtelomerase inhibitors or telomeric DNA binding compounds. For example,suitable alkylating agents include alkyl sulfonates, such as busulfan,improsulfan, and piposulfan; aziridines, such as a benzodizepa,carboquone, meturedepa, and uredepa; ethylenimines and methylmelamines,such as altretamine, triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide, and trimethylolmelamine; nitrogen mustardssuch as chlorambucil, chlornaphazine, cyclophosphamide, estramustine,iphosphamide, mechlorethamine, mechlorethamine oxide hydrochloride,melphalan, novembichine, phenesterine, prednimustine, trofosfamide, anduracil mustard; nitroso ureas, such as carmustine, chlorozotocin,fotemustine, lomustine, nimustine, and ranimustine.

Antibiotics used in the treatment of cancer include dactinomycin,daunorubicin, doxorubicin, idarubicin, bleomycin sulfate, mytomycin,plicamycin, and streptozocin. Chemotherapeutic antimetabolites includemercaptopurine, thioguanine, cladribine, fludarabine phosphate,fluorouracil (5-FU), floxuridine, cytarabine, pentostatin, methotrexate,and azathioprine, acyclovir, adenine β-1-D-arabinoside, amethopterin,aminopterin, 2-aminopurine, aphidicolin, 8-azaguanine, azaserine,6-azauracil, 2′-azido-2′-deoxynucleosides, 5-bromodeoxycytidine,cytosine β-1-D-arabinoside, diazooxynorleucine, dideoxynucleosides,5-fluorodeoxycytidine, 5-fluorodeoxyuridine, and hydroxyurea.

Chemotherapeutic protein synthesis inhibitors include abrin,aurintricarboxylic acid, chloramphenicol, colicin E3, cycloheximide,diphtheria toxin, edeine A, emetine, erythromycin, ethionine, fluoride,5-fluorotryptophan, fusidic acid, guanylyl methylene diphosphonate andguanylyl imidodiphosphate, kanamycin, kasugamycin, kirromycin, andO-methyl threonine. Additional protein synthesis inhibitors includemodeccin, neomycin, norvaline, pactamycin, paromomycine, puromycin,ricin, shiga toxin, showdomycin, sparsomycin, spectinomycin,streptomycin, tetracycline, thiostrepton, and trimethoprim. Inhibitorsof DNA synthesis, including alkylating agents such as dimethyl sulfate,mitomycin C, nitrogen and sulfur mustards, intercalating agents, such asacridine dyes, actinomycins, adriamycin, anthracenes, benzopyrene,ethidium bromide, propidium diiodide-intertwining, and agents, such asdistamycin and netropsin, can be used as part of the presently disclosedcancer treatments. Topoisomerase inhibitors, such as coumermycin,nalidixic acid, novobiocin, and oxolinic acid, inhibitors of celldivision, including colcemide, colchicine, vinblastine, and vincristine;and RNA synthesis inhibitors including actinomycin D, a-amanitine andother fungal amatoxins, cordycepin (3′-deoxyadenosine),dichlororibofuranosyl benzimidazole, rifampicine, streptovaricin, andstreptolydigin also can be combined with or incorporated into theparticles of the presently disclosed subject matter to provide asuitable cancer treatment.

Thus, current chemotherapeutic agents that can be used as part of or incombination with the presently describe NO-releasing particles include,adrimycin, 5-fluorouracil (5FU), etoposide, camptothecin, actinomycin-D,mitomycin, cisplatin, hydrogen peroxide, carboplatin, procarbazine,mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chjlorambucil,bisulfan, nitrosurea, dactinomycin, duanorubicin, doxorubicin,bleomycin, pilcomycin, tamoxifen, taxol, transplatimun, vinblastin, andmethotrexate, and the like.

As used herein, the term “antimicrobial agent” refers to any agent thatkills, inhibits the growth of, or prevents the growth of a bacteria,fungus, yeast, or virus. Suitable antimicrobial agents that can beincorporated into the presently disclosed NO-releasing particles to aidin the treatment or prevention of a microbial infection, include, butare not limited to, antibiotics such as vancomycin, bleomycin,pentostatin, mitoxantrone, mitomycin, dactinomycin, plicamycin andamikacin. Other antimicrobial agents include antibacterial agents suchas 2-p-sulfanilyanilinoethanol, 4,4′-sulfinyldianiline,4-sulfanilamidosalicylic acid, acediasulfone, acetosulfone, amikacin,amoxicillin, amphotericin B, ampicillin, apalcillin, apicycline,apramycin, arbekacin, aspoxicillin, azidamfenicol, azithromycin,aztreonam, bacitracin, bambermycin(s), biapenem, brodimoprim, butirosin,capreomycin, carbenicillin, carbomycin, carumonam, cefadroxil,cefamandole, cefatrizine, cefbuperazone, cefclidin, cefdinir,cefditoren, cefepime, cefetamet, cefixime, cefmenoxime, cefininox,cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotetan,cefotiam, cefozopran, cefpimizole, cefpiramide, cefpirome, cefprozil,cefroxadine, ceftazidime, cefteram, ceftibuten, ceftriaxone, cefuzonam,cephalexin, cephaloglycin, cephalosporin C, cephradine, chloramphenicol,chlortetracycline, ciprofloxacin, clarithromycin, clinafloxacin,clindamycin, clindamycin phosphate, clomocycline, colistin, cyclacillin,dapsone, demecicycline, diathymosulfone, dibekacin, dihydrostreptomycin,dirithromycin, doxycycline, enoxacin, enviomycin, epicillin,erythromycin, flomoxef, fortimicin(s), gentamicin(s), glucosulfonesolasulfone, gramicidin S, gramicidin(s), grepafloxacin, guamecycline,hetacillin, imipenem, isepamicin, josamycin, kanamycin(s),leucomycin(s), lincomycin, lomefloxacin, lucensomycin, lymecycline,meclocycline, meropenem, methacycline, micronomicin, midecamycin(s),minocycline, moxalactam, mupirocin, nadifloxacin, natamycin, neomycin,netilmicin, norfloxacin, oleandomycin, oxytetracycline,p-sulfanilylbenzylamine, panipenem, paromomycin, pazufloxacin,penicillin N, pipacycline, pipemidic acid, polymyxin, primycin,quinacillin, ribostamycin, rifamide, rifampin, rifamycin SV,rifapentine, rifaximin, ristocetin, ritipenem, rokitamycin,rolitetracycline, rosaramycin, roxithromycin, salazosulfadimidine,sancycline, sisomicin, sparfloxacin, spectinomycin, spiramycin,streptomycin, succisulfone, sulfachrysoidine, sulfaloxic acid,sulfamidochrysoidine, sulfanilic acid, sulfoxone, teicoplanin,temafloxacin, temocillin, tetracycline, tetroxoprim, thiamphenicol,thiazolsulfone, thiostrepton, ticarcillin, tigemonam, tobramycin,tosufloxacin, trimethoprim, trospectomycin, trovafloxacin,tuberactinomycin and vancomycin. Antimicrobial agents can also includeanti-fungals, such as amphotericin B, azaserine, candicidin(s),chlorphenesin, dermostatin(s), filipin, fungichromin, mepartricin,nystatin, oligomycin(s), perimycin A, tubercidin, imidazoles, triazoles,and griesofulvin.

V. METHODS OF TREATMENT

Accordingly, in some embodiments, the presently disclosed subject matterprovides a method for the delivery of nitric oxide to a subject, whichin some embodiments is intended to treat a disease or condition in asubject in need of treatment thereof. In some embodiments, the presentlydisclosed subject matter provides a method for the targeted delivery ofnitric oxide to a specific site in a subject. Such a site can bespecific cells, tissues or organs. Thus, the presently disclosed subjectmatter provides a method for treating cancer, cardiovascular diseases,and microbial infections; for the inhibition of platelet aggregation andplatelet adhesion caused by the exposure of blood to a medical device;for treating pathological conditions resulting from abnormal cellproliferation; transplantation rejections, autoimmune, inflammatory,proliferative, hyperproliferative, vascular diseases; for reducing scartissue or for inhibiting wound contraction, including the prophylacticand/or therapeutic treatment of restenosis by administering the nitricoxide donor optionally in combination with at least one additionaltherapeutic agent. The presently disclosed subject matter also providesa method for treating inflammation, pain, fever, gastrointestinaldisorders, respiratory disorders, sexual dysfunctions, and sexuallytransmitted diseases.

V.A. Subjects

In some embodiments, the methods of the presently disclosed subjectmatter can be useful for treatment of a subject, as defined herein. Thesubject treated in the presently disclosed subject matter in its manyembodiments is a human subject, although it is to be understood that theprinciples of the presently disclosed subject matter indicate that thepresently disclosed subject matter is effective with respect to allvertebrate species, including mammals, which are intended to be includedin the term “subject”. In this context, a mammal is understood toinclude any mammalian species in which treatment is desirable,particularly agricultural and domestic mammalian species.

Accordingly, the term “subject” as used herein, refers to anyinvertebrate or vertebrate species. The methods of the presentlydisclosed subject matter are particularly useful in the treatment ofwarm-blooded vertebrates. Thus, the presently disclosed subject matterconcerns mammals and birds. More particularly, provided is the treatmentand/or diagnosis of mammals, such as humans, as well as those mammals ofimportance due to being endangered (such as Siberian tigers), ofeconomical importance (animals raised on farms for consumption byhumans) and/or social importance (animals kept as pets or in zoos) tohumans, for instance, carnivores other than humans (such as cats anddogs), swine (pigs, hogs, and wild boars), ruminants (such as cattle,oxen, sheep, giraffes, deer, goats, bison, and camels), and horses. Alsoprovided is the treatment of birds, including the treatment of thosekinds of birds that are endangered, kept in zoos, as well as fowl, andmore particularly domesticated fowl, e.g., poultry, such as turkeys,chickens, ducks, geese, guinea fowl, and the like, as they also are ofeconomical importance to humans. Thus, provided is the treatment oflivestock, including, but not limited to, domesticated swine (pigs andhogs), ruminants, horses, poultry, and the like.

V.B. Formulations

The presently disclosed therapeutic compositions, in some embodiments,comprise a composition that includes a presently disclosed nitricoxide-releasing nanoparticle and a pharmaceutically acceptable carrier.Suitable compositions include aqueous and non-aqueous sterile injectionsolutions that can contain antioxidants, buffers, bacteriostats,bactericidal antibiotics and solutes that render the formulationisotonic with the bodily fluids of the intended recipient; and aqueousand non-aqueous sterile suspensions, which can include suspending agentsand thickening agents.

In some embodiments, the presently disclosed therapeutic compositionscomprise an additional therapeutic agent in combination with the nitricoxide-releasing nanoparticles, wherein the additional therapeutic agenthas additional desired therapeutic properties or enhances thetherapeutic properties of the nitric oxide-releasing nanoparticles. Theadditional therapeutic agent can be administered in the same or adifferent therapeutic composition. Thus, the term “in combination” canrefer to the administration of active agents in a single composition orin one or more separate compositions.

The compositions used in the presently disclosed methods can take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and can contain formulatory agents, such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The therapeutic compositions can be presented in unit-dose or multi-dosecontainers, for example sealed ampoules and vials, and can be stored ina frozen or freeze-dried (lyophilized) condition requiring only theaddition of sterile liquid carrier immediately prior to use.

For oral administration, the compositions can take the form of, forexample, tablets or capsules prepared by a conventional technique withpharmaceutically acceptable excipients, such as binding agents (e.g.,pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose orcalcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talcor silica); disintegrants (e.g., potato starch or sodium starchglycollate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets can be coated by methods known in the art. For example, atherapeutic agent can be formulated in combination withhydrochlorothiazide, and as a pH stabilized core having an enteric ordelayed release coating which protects the therapeutic agent until itreaches the target organ.

Liquid preparations for oral administration can take the form of, forexample, solutions, syrups or suspensions, or they can be presented as adry product for constitution with water or other suitable vehicle beforeuse. Such liquid preparations can be prepared by conventional techniqueswith pharmaceutically acceptable additives, such as suspending agents(e.g., sorbitol syrup, cellulose derivatives or hydrogenated ediblefats); emulsifying agents (e.g., lecithin or acacia); non-aqueousvehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionatedvegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The preparations also cancontain buffer salts, flavoring, coloring and sweetening agents asappropriate. Preparations for oral administration can be suitablyformulated to give controlled release of the active compound. For buccaladministration the compositions can take the form of tablets or lozengesformulated in conventional manner.

The compounds also can be formulated as a preparation for implantationor injection. Thus, for example, the compounds can be formulated withsuitable polymeric or hydrophobic materials (e.g., as an emulsion in anacceptable oil) or ion exchange resins, or as sparingly solublederivatives (e.g., as a sparingly soluble salt). The compounds also canbe formulated in rectal compositions (e.g., suppositories or retentionenemas containing conventional suppository bases, such as cocoa butteror other glycerides), creams or lotions, or transdermal patches.

Pharmaceutical formulations also are provided which are suitable foradministration as an aerosol by inhalation. These formulations comprisea solution or suspension of a NO-releasing particle described herein.The desired formulation can be placed in a small chamber and nebulized.Nebulization can be accomplished by compressed air or by ultrasonicenergy to form a plurality of liquid droplets or solid particlescomprising the NO-releasing particles. For example, the presentlydisclosed NO-releasing particles can be administered via inhalation totreat bacterial infections related to cystic fibrosis. Cysticfibrosis-related bacterial infections include, but are not limited to,Pseudomonas aeruginosa (P. aeruginosa) infections.

V.C. Doses

The term “effective amount” is used herein to refer to an amount of thetherapeutic composition (e.g., a composition comprising a nitricoxide-releasing particle) sufficient to produce a measurable biologicalresponse. Actual dosage levels of active ingredients in an activecomposition of the presently disclosed subject matter can be varied soas to administer an amount of the active compound(s) that is effectiveto achieve the desired response for a particular subject and/orapplication. The selected dosage level will depend upon a variety offactors including the activity of the composition, formulation, theroute of administration, combination with other drugs or treatments,severity of the condition being treated, and the physical condition andprior medical history of the subject being treated. Preferably, aminimal dose is administered, and dose is escalated in the absence ofdose-limiting toxicity to a minimally effective amount. Determinationand adjustment of an effective dose, as well as evaluation of when andhow to make such adjustments, are known to those of ordinary skill inthe art of medicine.

For administration of a composition as disclosed herein, conventionalmethods of extrapolating human dosage based on doses administered to amurine animal model can be carried out using the conversion factor forconverting the mouse dosage to human dosage: Dose Human per kg=DoseMouse per kg×12. See Freireich et al., Cancer Chemother Rep. 50, 219-244(1966). Drug doses also can be given in milligrams per square meter ofbody surface area because this method rather than body weight achieves agood correlation to certain metabolic and excretionary functions.Moreover, body surface area can be used as a common denominator for drugdosage in adults and children as well as in different animal species.See Freireich et al., Cancer Chemother Rep. 50, 219-244 (1966). Briefly,to express a mg/kg dose in any given species as the equivalent mg/sq mdose, multiply the dose by the appropriate km factor. In an adult human,100 mg/kg is equivalent to 100 mg/kg×37 kg/sq m=3700 mg/m².

For additional guidance regarding formulation and dose, see U.S. Pat.Nos. 5,326,902; 5,234,933; PCT International Publication No. WO93/25521; Berkow et al., The Merck Manual of Medical Information, Homeed., Merck Research Laboratories: Whitehouse Station, N.J. (1997);Goodman et al., Goodman & Gilman's the Pharmacological Basis ofTherapeutics, 9th ed. McGraw-Hill Health Professions Division: New York(1996); Ebadi, CRC Desk Reference of Clinical Pharmacology, CRC Press,Boca Raton, Fla. (1998); Katzunq, Basic & Clinical Pharmacology, 8th ed.Lange Medical Books/McGraw-Hill Medical Pub. Division: New York (2001);Remington et al., Remington's Pharmaceutical Sciences, 15th ed. MackPub. Co.: Easton, Pa. (1975); and Speight et al., Avery's DrugTreatment: A Guide to the Properties, Choice, Therapeutic Use andEconomic Value of Drugs in Disease Management, 4th ed. AdisInternational: Auckland/Philadelphia (1997); Dutch et al., Toxicol.Lett., 100-101, 255-263 (1998).

V.D. Routes of Administration

Suitable methods for administering to a subject a composition of thepresently disclosed subject matter include, but are not limited to,systemic administration, parenteral administration (includingintravascular, intramuscular, intraarterial administration), oraldelivery, buccal delivery, subcutaneous administration, inhalation,intratracheal installation, surgical implantation, transdermal delivery,local injection, and hyper-velocity injection/bombardment. Whereapplicable, continuous infusion can enhance drug accumulation at atarget site (see, e.g., U.S. Pat. No. 6,180,082).

The particular mode of drug administration used in accordance with themethods of the presently disclosed subject matter depends on variousfactors, including but not limited to the agent and/or carrier employed,the severity of the condition to be treated, and mechanisms formetabolism or removal of the active agent following administration.

VI. COMPOSITIONS CONTAINING NO-RELEASING PARTICLES

In some embodiments, the NO-releasing particles can be incorporated intopolymeric films. Such incorporation can be through physically embeddingthe particles into polymer surfaces, via electrostatic association ofparticles onto polymeric surfaces, or by covalent attachment ofparticles onto reactive groups on the surface of a polymer.Alternatively, the particles can be mixed into a solution of liquidpolymer precursor, becoming entrapped in the polymer matrix when thepolymer is cured. Polymerizable groups can also be used to functionalizethe exterior of the particles, whereupon, the particles can beco-polymerized into a polymer during the polymerization process.Suitable polymers into which the NO-releasing particles can beincorporated include polyolefins, such as polystyrene, polypropylene,polyethylene, polytetrafluoroethylene, and polyvinylidene, as well aspolyesters, polyethers, polyurethanes, and the like. In particular,polyurethanes can include medically segmented polyurethanes. Ageneralized structure for a medically segmented polyurethane is shown inFIG. 9A. Such polyurethanes can include hard segments, i.e., moietiesthat are relatively rigid, and soft segments, i.e., moieties having moredegrees of freedom that can exist in a number of alternate,inter-converting conformations. Medically segmented polyurethanes canalso include one or more expander moieties, such as alkylene chains,that add additional length or weight to the polymer. Such polyurethanesare also generally non-toxic. One example of a medically segmentedpolyurethane is TECOFLEX®. See FIG. 9B.

Polymeric films containing NO-releasing particles can be used to coat avariety of articles, particularly surgical tools, biological sensors,and medical implants to prevent platelet adhesion, to prevent bacterialinfection, to act as a vasodilator. These articles can be of use invascular medical devices, urological medical devised, biliary medicaldevices, gastrointestinal medical devices, medical devices adapted forplacement at surgical sites, and medical devices adapted for placementon skin wounds or openings. Thus, the polymers can be used to coatarterial stents, guide wires, catheters, trocar needles, bone anchors,bone screws, protective platings, hip and joint replacements, electricalleads, biosensors, probes, sutures, surgical drapes, wound dressings,and bandages.

In some embodiments, the device being coated can have a metallicsurface, such as, for example, stainless steel, nickel, titanium,aluminum, copper, gold, silver, platinum, and combinations thereof. Insome embodiments, the films or polymers containing the NO-releasingparticles can be used to coat non-metallic surfaces, such as glass orfiber (e.g., cloth or paper)

Additionally, polymers containing NO-releasing particles can be used toform the devices, themselves. For example, the polymers can be fashionedinto storage bags for blood or tissue or as wound dressings.

Further, the NO-releasing particles can be incorporated into detergents,such as, but not limited to, anti-microbial soaps. For example,NO-release in particles embedded in bar soaps can be triggered bycontact with water and/or a drop in pH upon use. As the outer surface ofthe bar is eroded or dissolved, additional particles within the barsurface become exposed for subsequent uses of the bar. NO-releasingparticles also can be suspended in liquid soaps. Such soaps ordetergents can be used for personal hygiene or to provide anti-microbialtreatments for fibers. Such soaps or detergents can also be used totreat household surfaces or any surface in a hospital or other medicalenvironment that may be exposed to microbes such as bacteria, fungi orviruses.

EXAMPLES

The following Examples have been included to provide guidance to one ofordinary skill in the art for practicing representative embodiments ofthe presently disclosed subject matter. In light of the presentdisclosure and the general level of skill in the art, those of skill canappreciate that the following Examples are intended to be exemplary onlyand that numerous changes, modifications, and alterations can beemployed without departing from the scope of the presently disclosedsubject matter.

Example 1 Synthesis of Amine Functionalized Gold Nanoparticles

Gold nanoparticles were functionalized with amines in a two-step processby first place exchanging Br-functionalized thiol ligands onto the goldnanoparticle core with subsequent addition of amine by a reaction withBr. See FIG. 2. Sample ¹H NMR spectra were acquired for each step of thesynthesis, as presented in FIG. 10.

More particularly, gold nanoparticles were synthesized by the Brustmethod, via the reaction of hydrogen tetrachloroaurate salt withhexanethiol in the presence of sodium borohydride. See Hostetler, M. I.,et al., Langmuir, 14, 17-30 (1998), After 30 min, the reaction wasquenched with water. The nanoparticles were collected by filtration andwashed with acetonitrile, then functionalized with bromo-terminatedalkanethiols by the place exchange method. See Hostetler, M. I., et al.,Langmuir, 15, 3782-3789 (1999).

The incoming bromo-terminated ligand (11-Bromo-1-undecanethiol assynthesized in Example 2, described herein below), see Troughton B. B.,et al., Langmuir, 4, 365-385 (1988), was added (3:1 ratio of bromo- tomethyl-terminated alkanethiol) to a solution of gold nanoparticles inmethylene chloride and stirred for 30 min. The solvent was removed byrotary evaporation, and the gold nanoparticles were purified withacetonitrile. The extent of ligand exchange, monitored by NMR, wascontrolled by varying the reaction time and/or concentration ofbromo-alkanethiol. The bromo-functionalized gold nanoparticles were thendissolved in toluene or methylene chloride and reacted withethylenediamine, butylamine, hexanediamine, or diethylenetriamine. Thedisappearance of the —CH₂Br peak in the NMR spectra of thefunctionalized nanoparticles indicated the completion of the reaction(See FIG. 10). The amine-functionalized gold nanoparticles were thensuspended in a solution of methanol and sodium methoxide base andpressurized to 5 atm NO for 3 days with constant stirring to facilitatethe synthesis of diazeniumdiolate NO donors. TheN-diazeniumdiolate-modified monolayer protected clusters (MPCs) werefiltered, washed with excess methanol, and stored at −4° C. until use.

The size and stability of the MPC gold nanoparticles were characterizedusing thermal gravimetric analysis (TGA), UV-Vis spectroscopy, andtransmission electron microscopy (TEM). The organic content ofhexanediamine-modified gold nanoparticles was determined to beapproximately 22%, a value consistent with previous reports forhexanethiol-MPCs composed of 140 gold atoms (core) protected by 53 thiolligands. See Hostetler, M. I., et al., Langmuir, 14, 17-30 (1998).

Because NO is highly reactive and might disrupt gold sulfur bonds, seeHrabie, J. A. and Keefer, L. K., Chemical Reviews, 102, 1135-1154(2002), the stability of the hexanethiol-MPCs after exposure to highpressures of NO was evaluated using TGA and UV-Vis spectroscopy toensure that the conditions necessary for diazeniumdiolate formation didnot compromise nanoparticle integrity. Both the organic content of thenanoparticles (as studied by TGA) and the UV-Vis spectra remained thesame following NO exposure indicating negligible influence on monolayerstability. Transmission electron microscopy images further confirmedthat the core diameter of the nanoparticles remained constant (2.1±0.9nm) regardless of amine derivatization or diazeniumdiolate formation.These studies suggest that the structural integrity of the MPC goldnanoparticles was not compromised by the conditions necessary tosynthesize the NO donor and Introduce NO-release capability.

Example 2 11-Bromo-1-Undecanethiol Synthesis

11-Bromo-1-undecanethiol was synthesized in two steps (see FIG. 11).First, 11-bromo-1-undecene (5.0 g) was converted to a thioacetate byreacting with AiBN (1.5 g) and thioacetic acid (10 mL) in toluene (50mL). The reaction was run under Ar and refluxed for 2 h. The solutionwas washed with excess water and the toluene removed by rotaryevaporation. The thioacetate was converted into a thiol by exposing the11-bromo-1-undecanethioacetate to dry HCI. Acetyl chloride (6 mL) wasadded dropwise to dry methanol in an ice bath under Ar. The solution wasallowed to warm to room temperature and the reaction progressed forapproximately 6 h. Methylene chloride and water were added and themethylene chloride layer was washed several times with water. Thesolvent was removed by rotary evaporation.

Example 3 General Procedure for Measuring Nitric Oxide Release

Nitric oxide release of the presently disclosed NO-releasing particleswas measured according to the following general procedure. Referring nowto FIG. 12, a predetermined volume of phosphate buffer solution (PBS)(pH 7.4, 37° C.) was disposed in a receptacle, e.g., a round-bottomedflask. The receptacle was sealed, leaving an inlet for nitrogen gas andan outlet for a mixture of nitrogen and nitric oxide. The outlet was influid communication with a chemiluminescence nitric oxide analyzer. Analiquot of a solution containing a diazeniumdiolated species wasinjected into the PBS buffer. The chemiluminescence nitric oxideanalyzer measured the amount of NO that reacted with ozone (O3) to formexcited NO₂*, which emitted electromagnetic radiation (hv) as shown inScheme 2.

Example 4 Measurement of Nitric Oxide Release from Amine-DerivatizedMonolayer Protected Gold Nanoparticles

Nitric oxide release was measured in phosphate buffered saline solutionat physiological temperature and pH using a Sievers NOA™chemiluminescence nitric oxide analyzer (Boulder, Colo., United Statesof America). As presented in Table 1, below and in FIG. 13, theNO-release for diazeniumdiolate-modified gold nanoparticles was tunableby varying the number and/or the chemical structure of the substitutedamine ligands. A schematic showing the release of nitric oxide from afunctionalized monolayer protected cluster (MPC) gold nanoparticle isshown in FIG. 14.

TABLE 1 Nitric Oxide Release Properties of Amine-Derivatized MonolayerProtected Gold Nanoparticles. % Half-life Release Total NO Ligand Amine(min) Longevity (min) (pmol/mg) Hexane —  2  55 400 Butylamine 21 15  602,000 Ethylenediamine 14 78 200 9,750 Ethylenediamine 21 88 300 19,300Hexanediamine 21 68 600 87,000 Diethylenetriamine 21 63 360 38,000

Example 5 Results from NO-Releasing Particles Comprising MonolayerProtected Gold Nanoparticles

Referring once again to Table 1 and FIG. 13, increasing theconcentration of ethylenediamine ligand from 14 to 21% led to acorresponding increase in total NO release (9750 to 19,300 pmol NO/mgMPC) and NO release duration (from 200 to 300 min). Without being boundto any particular theory of operation, it is suggested that the elevatedNO release is attributed to enhanced NO-donor formation due to a largerconcentration of amines. A small amount of NO (400 pmol/mg) also wasmeasured from the hexanethiol MPC controls. This NO release wasnegligible at periods greater than 5 min, suggesting that a small amountof NO likely intercalates within the hydrophobic alkyl chains under theconditions necessary for diazeniumdiolate synthesis (5 atm NO), but suchNO is rapidly released upon solution immersion.

The diazeniumdiolate-modified MPCs also released low levels of NO undera warm (37° C.) stream of nitrogen gas, suggesting a possible thermaldissociation mechanism. The level of NO release, however, was greater inbuffer, suggesting that the N-diazeniumdiolate-modified nanoparticlesundergo both proton driven and thermal dissociation. Thediazeniumdiolate-modified MPCs retained full NO release characteristicswhen stored under nitrogen at −4° C. for up to 14 days (the longestperiod investigated).

The NO release from diazeniumdiolate-modified MPCs also was tunable byvarying the amine precursor structure. Increasing the length of thealkyl chain separating the nitrogens from two to six methylene units ledto an increase in the total amount of NO released (see Table 1 and FIGS.13, d and f) (19,300 to 87,000 pmol NO/mg MPC for ethylenediamine- andhexanediamine-modified MPCs, respectively), suggesting a NOrelease/diazeniumdiolate structure relationship.

Indeed, the half-life data (Table 1) show that separating the aminesresults in a more rapid release of NO as well, analogous to thedissociation behavior reported for small molecule diazeniumdiolates. SeeHrabie, J. A., et al., J. Org. Chem., 58, 1472-1476 (1993); Davies, K.M., et al., J. Am. Chem. Soc., 123, 5473-5481 (2001).

The total amount of NO released from diethylenetriamine-modified MPCs(38,000 pmol NO/mg) was between that measured for ethylenediamine- andhexanediamine-modified MPCs. The presence of an additional secondaryamine in diethylenetriamine likely accounts for increased NO donorformation (and release capability) relative to ethylenediamine, eventhough the length of the alkyl chain separating the nitrogens remainsshort (two methylene units).

Butylamine-modified MPCs, a secondary monoamine derivative, werecharacterized by the lowest total NO release of all the amine-modifiedMPCs studied. Diazeniumdiolate formation is facilitated by theadditional amine. See Hrabie, J. A., et al., J. Org. Chem., 58,1472-1476 (1993); Davies, K. M., et al., J. Am. Chem. Soc., 123,5473-5481 (2001). Notably, the diazeniumdiolate conversion efficiencyfor the amine-modified MPCs was calculated to be less than 1%,regardless of amine structure.

Example 6 Preparation of Nitric Oxide-Releasing Dendrimers

Polypropylenimine hexadecaamine dendrimer (DAB-Am-16, available fromAldrich Chemical Company, Milwaukee, Wis., United States of America)(see FIG. 15) was charged at 5-atm nitric oxide for three days in thepresence of sodium methoxide (NaOMe). This procedure yielded 0.74 molesnitric oxide/mole dendrimer (2.3% conversion) and 2.3×10⁸ moles nitricoxide released.

Polypropylenimine tetrahexacontaamine dendrimer (DAB-Am-64, availablefrom Aldrich Chemical Company, Milwaukee, Wis., United States ofAmerica) (see FIG. 16) was charged at 5-atm nitric oxide for three daysin the presence of NaOMe. This procedure yielded 4.94 moles nitricoxide/mole dendrimer (3.9% conversion) and 1.18×10⁸ moles nitric oxidereleased.

DAB-C7-16 (see Scheme 4 below) was charged at 5-atm nitric oxide forthree days In the presence of NaOMe/MeOH (Scheme 5). This procedureyielded 12 moles NO/mole dendrimer (37.9% conversion) and 3.74×10⁻⁷moles NO released.

DAB-C7-64 was charged at 5-atm nitric oxide for three days in thepresence of NaOMe/MeOH. This procedure yielded 45 moles NO/moledendrimer (35.6% conversion) and 1.48×10⁻⁷ moles NO released.

A graph showing nitric oxide release versus time for DAB-C7-16NaOMe/MeOH is shown in FIG. 17. Likewise, a graph showing nitric oxiderelease versus time for DAB-C7-64 NaOMe/MeOH is shown in FIG. 18.

DAB-Ac-16 (Scheme 6) was charged at 5-atm nitric oxide for three days inthe presence of NaOMe. This procedure yielded 0.039 moles NO/moledendrimer (0.12% conversion) and 4.95×10¹⁰ moles NO released.

DAB-Ac-64 was charged at 5-atm nitric oxide for three days in thepresence of NaOMe. This procedure yielded 0.22 moles NO/mole dendrimer(0.17% conversion) and 3.75×10⁻¹⁰ moles NO released.

DAB-Pro-16 (Scheme 7) was charged at 5-atm nitric oxide for three daysin the presence of NaOMe. This procedure yielded 42 moles NO/moledendrimer (130% conversion) and 1.92×10⁻⁷ moles NO released.

DAB-Pro-64 was charged at 5-atm nitric oxide for three days in thepresence of NaOMe. This procedure yielded 480 moles NO/mole dendrimer(377% conversion) and 4.79×10⁻⁷ moles NO released.

Example 7 Measurement of Nitric Oxide Release from Amine-DerivatizedDendrimers

NO release from amine-derivatized dendrimers synthesized as described inExample 6 was measured according to the procedure outlined in Example 1Results are summarized below in Table 2.

TABLE 2 Summary of Nitric Oxide Release from Amine DerivatizedDendrimers NO Released moles Diazeniumdiolated (mmol T_(1/2) NO/molAmine Species NO/g) (min) dendrimer Structure DAB-Ac-16 0.016 1.4 0.04capped DAB-Ac-64 0.02 2.5 0.22 DAB-Am-16 0.44 12 0.74 primary DAB-Am-640.69 29 4.94 DAB-C7-16 3.4 80 12 secondary DAB-C7-64 3.2 90 45DAB-Pro-16 13 150 42 secondary DAB-Pro-64 36 117 480

Example 8 Measurement of Nitric Oxide Release from DiazeniumdiolatedMaterials

NO release from a variety of NO-releasing materials was measuredaccording to the procedure outlined in Example 3. Results are summarizedbelow in Table 3. The diazeniumdiolated fumed silica particles wereprepared as described in Example 9, below, grafting the fumed silicasurface to N-(6-aminohexyl)-3-aminopropyltrimethoxysilane, followed bydiazeniumdiolation of the secondary amine with NO gas.

TABLE 3 Summary of Nitric Oxide Release from Diazeniumdiolated MaterialsDiazeniumdiolated Species NO Released (mmol NO/g) tv2 (min) Proteins(Bovine serum 0.54 7.2E4. albumin (BSA) Fumed Silica (2N[6]-N₂0₂ 0.56 43Sol-gels (20% AHAP3) 0.24 45 Polymethacrylate (C2-ED) 0.94 60

Example 9 Synthesis Route to NO-Releasing Silica Particles

Referring now to FIG. 19, NO-releasing silica particles with a particlesize ranging from about 200 nm to about 300 nm are prepared followingthe method described by Zhang, H. et al., J. Am. Chem. Soc., 125, 5015(2003).

Example 10 Synthesis of Silica Based on Co-Condensation of NO DonorPrecursors

Reagents and Materials:

Tetraethyl orthosilicate (TEOS), tetramethylsilane (TMS), and sodiummethoxide (NaOMe) were purchased from Fluke (Buchs, Switzerland).Silanes including (aminoethylaminomethyl)phenethyltrimeth-oxysilane(AEMP3), N-(6-aminohexyl)aminopropyltrimethoxysilane (AHAP3),N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (AEAP3), andN-[3-(trimeth-oxysilyl)propyl]diethylenetriamine (DET3) were purchasedfrom Gelest (Tullytown, Pa., United States of America).N,N-Dimethylformamide (DMF) was purchased from Sigma Chemical Company(St. Louis, Mo., United States of America). Methanol (MeOH), ethanol(EtOH), toluene, and ammonia solution (NH₄OH, 30 wt % in water) werepurchased from Fisher Scientific (Fair Lawn, N.J., United States ofAmerica). Nitric oxide (NO, 99.5%), argon (Ar), and nitrogen (N₂) gaseswere obtained from AGA Gas (Maumee, Ohio, United States of America) orNational Welders Supply (Raleigh, N.C., United States of America). Othersolvents and chemicals were analytical-reagent grade and used asreceived. A Millipore Milli-Q UV Gradient A10 System (MilliporeCorporation, Bedford, Mass., United States of America) was used topurify distilled water to a final resistivity of 18.2 MΩ-2·cm and atotal organic content≦6 ppb.

Synthesis of Nitric Oxide-Releasing Silica Nanoparticles:

Silane solutions were prepared by mixing 2.78 mmol (620 μL) of TEOS withdifferent concentrations of AEAP3, AHAP3, AEMP3, or DET3 (0-0.70 mmolcorresponding to 0-20 mol %, balance TEOS) for 10 min. The silanesolution was then combined with 22 mL of EtOH and 6 mL of ammonia (30 wt% in water), and vigorously stirred for 30 min under ambient conditions.The white precipitate was collected by centrifugation (5000 rpm, 5 min),washed with EtOH copiously, and dried under vacuum overnight.

The resulting amine-functionalized silica was resuspended in 18 mL ofDMF and 2 mL of MeOH in the presence of NaOMe (0.32-0.70 mmol; adding anequimolar amount of NaOMe corresponding to the secondary amine contentof silica composites) and placed in 10 mL-vials equipped with a stirbar. The vials were placed in a Parr bottle (200 mL), connected to anin-house NO reactor, and flushed with Ar six times to remove oxygen inthe suspension. The reaction bottle was then charged with NO to 5 atmand sealed for 3 d while stirring. The NO gas was purified over KOHpellets for 2 h to remove trace NO degradation products. Prior toremoving the silica particles, unreacted NO was purged from the chamberwith Ar. The N-diazeniumdiolate-modified silica particles wererecollected by centrifugation at 5000 rpm for 5 min, washed copiouslywith ethanol, dried under ambient conditions for 1 h, and stored in asealed container at −20° C. until used

Example 11 Characterization of Functionalized Silica

Solid-state cross polarization/magnetic angle spinning (CP/MAS) ²⁹Sinuclear magnetic resonance (NMR) spectra were obtained at 293 K on aBruker 360 MHz DMX spectrometer (Billerica, Mass., United States ofAmerica) equipped with wide-bore magnets (triple axis pulsed fieldgradient double resonance probes). Silica composite particles (0, 10,13, and 17 mol % AEAP3, balance TEOS) were packed into 4 mm rotors(double resonance frequency of 71.548 MHz) and spun at a speed of 8.0kHz. The chemical shifts were determined in ppm relative to a TMSexternal standard.

For atomic force microscopy (AFM) imaging, the silica particles weresuspended in toluene, deposited on a freshly cleaved mica surface, anddried under ambient conditions for 3 h. Contact mode AFM images wereobtained in air using a Molecular Force Probe 3D Atomic Force Microscope(Asylum Research; Santa Barbara, Calif., United States of America)controlled with a MFP-3D software running under Igor Pro (Wavemetrics;Lake Oswego, Oreg., United States of America). Triangular siliconnitride cantilevers with a nominal spring constant of 0.12 N/m⁻¹ andresonance frequency of 20 kHz (Veeco; Santa Barbara, Calif., UnitedStates of America) were used to acquire height/topography images at ascan rate of 0.5 Hz.

Nitric oxide release profiles of the N-diazeniumdiolate-modified silicananoparticles were measured in deoxygenated phosphate-buffered saline(PBS, 0.01 M; 37° C.) at a pH 3.3, 4.3, 5.3, 6.0, 7.4, and 9.5 using aSievers NOA 280i chemiluminescence nitric oxide analyzer (Boulder,Colo., United States of America). Nitric oxide released from the silicawas transported to the analyzer by a stream of N₂ (200 mL/min) passedthrough the reaction cell. The instrument was calibrated with air (0 ppmNO) passed through a zero filter, and 24.1 ppm of NO standard gas(balance N₂, purchased from AGA Gas).

The surface area and pore volume of the silica were determined vianitrogen adsorption/desorption isotherms (see, Huh, S., et al., Chem.Mater., 15, 4247-4256 (2003)) collected with a Beckman Coulter SA3100Surface Area and Pore Size Analyzer (Fullerton, Calif., United States ofAmerica). The surface area and pore volume were calculated using theBrunauer-Emmett-Teller (BET) and Barrett-Joyner-Halenda (BJH) methods.Prior to the measurements, all silica samples were degassed at 200° C.for 3 h.

Example 12 Physical Characteristics of NO-Release Silica NanoparticlesBased on Co-Condensation of NO Donor Precursors

The size of silica nanoparticles was tunable by varying the type andconcentration of aminoalkoxysilane used. Contact mode atomic forcemicroscope (AFM) images of silica spheres having different silanecompositions are shown in FIGS. 20A-20E. The diameter of control (TEOSonly) silica particles was 250±20 nm. Altering the TEOS solution toinclude 10 mol % AHAP3 decreased the diameter of the particles to 20±2nm. Silica particles prepared from AEAP3 and TEOS were roughly twice aslarge (d=500±45 nm) than controls. As the mol % of AEAP3 was increasedfrom 10 to 17 mol % (balance TEOS), the diameter of the particledecreased to 92±16 nm, revealing a pseudo-linear relationship betweensilica size and aminoalkoxysilane concentration (FIG. 20F). Similartrends in size were observed for each aminoalkoxysilane system studied.The size of the particles was not altered after N-diazeniumdiolatesynthesis, indicating that the structural integrity of the silicaparticles was not compromised by the conditions necessary to form the NOdonor and introduce NO release capability.

As shown in FIGS. 21A-21C, solid-state ²⁹Si nuclear magnetic resonance(NMR) was used to confirm the incorporation of aminoalkoxyfunctionalities within the silica network and to determine the surfacecoverage (SC) of such ligands. Cross polarization and magic anglespinning (CP/MAS) techniques were employed to increase the signalresolution and sensitivity. Control and amine-functionalized silicaparticles prepared from 0 to 17 mol % AEAP3 (balance TEOS) wereanalyzed. For TEOS control silica, three distinct peaks in the ²⁹Si NMRspectrum were observed at −90, −101, and −109 ppm, respectively,representative of Q² (geminal silanol; —O₂Si(OH)₂), Q³ (single silanol;—O₃Si(OH)), and Q⁴ (siloxane; —O₄Si) silicons. See Huh, S., et al.,Chem. Mater., 15, 4247-4256 (2003); and Albert, K., and Bayer, E. J., J.Chromatogr., 544, 345-370 (1991). For the aminoalkoxysilane-modifiedsilica particles, five peaks were observed in the spectra, indicatingthree additional silicon chemical environments (graphs b-d in FIG. 21A).The peaks at chemical shifts of approximately −52 and −65 ppm arerepresentative of silicon connected to T² (—O₂Si(OH)R) and T³ (—O₃SiR)structures, respectively (where R is an aminoethylaminopropyl group).See Huh, S., et al., Chem. Mater., 15, 4247-4256 (2003); and Albert, K,and Bayer, E. J., J. Chromatogr., 544, 345-370 (1991). The presence ofT¹¹ bands suggests the existence of covalent linkages betweenaminoalkoxy groups and the silica backbone. The resonance linesrepresenting Q², Q³, and Q⁴ were also assigned in the expectedpositions. As the AEAP3 content was increased from 10 to 17 mol %, thesurface coverage of aminoalkoxy ligands [SC=(T²+T³)/(T²+T³+Q²+Q³); SeeHuh, S., et al., Chem. Mater., 15, 4247-4256 (2003); and Radu, D. R., etal., J. Am. Chem. Soc., 126, 1640-1641 (2004)]. Increased from 21 to 37%correspondingly. See FIG. 21C. Of note, the quantitative analysis ofthese structures is complicated because the intensity of each peakdepends on the efficiency of cross polarization and the protonrelaxation time. See Bruch, M. D., and Fatunmbi, H. O., J. Chromatogr.A, 1021, 61-70 (2003).

The surface area and pore volume of the silica nanoparticles wereevaluated via nitrogen adsorption-desorption isotherms, as describedpreviously. See Huh, S. et al., Chem. Mater., 15, 4247-4256 (2003). Asexpected, the amine-functionalized silica proved to be nonporous withsurface areas (S_(BET)) of 10-20 m², g⁻¹ and pore volumes (V_(p)) of0.02-0.06 mL·g⁻¹ (at p/p₀=0.98).

Example 13 Results of NO-Release Silica Nanoparticles Based onCo-Condensation of NO Donor Precursors

NO release characteristics including the total amount of NO (t[NO]),half-life of NO release (t_(1/2)), maximum flux of NO release (NO]_(m)),and time necessary (t_(m)) to reach [NO]_(m) were evaluated as afunction of aminoalkoxysilane structure and amount. The results aresummarized in Table 4, below.

TABLE 4 NO Release Properties of Silica Particles Prepared based on theCo-condensation of NO Donor Precursors^(a) Ligand Mol t[NO] t½ [NO]_(m)t_(m) Type % (nmol/mg) (h) (ppb/mg) (h) AEP3 10 145 ± 10 12 ± 4   14 ± 38 ± 1 AEP3 13 392 ± 15   6 ± 1.5 92 ± 5 4 ± 1 AEP3 17 600 ± 25 3.4 ± 0.4140 ± 10 2.1 ± 0.3 AHAP3 10 380 ± 20 0.85 ± 0.05 370 ± 10 0.35 ± 0.05AEMP3 10 53 ± 3 6.0 ± 0.2 10 ± 2 0.12 ± 0.01 AEMP3 13 81 ± 3 6.5 ± 0.322 ± 2 0.10 ± 0.01 AEMP3 17 118 ± 5  5.7 ± 0.5 32 ± 2 0.11 ± 0.02 AEMP320 170 ± 10 5.4 ± 0.3 40 ± 3 0.11 ± 0.01 DET3 10 120 ± 5  4.0 ± 0.2 22 ±2 1.6 ± 0.1 ^(a)n is at least 3.

The NO release was measured in phosphate buffered saline (PBS) solutionat physiological temperature (37° C.) and pH (7.4) using achemiluminescence nitric oxide analyzer. See Beckman, J. S., and Conger,K. A., Methods Companion Methods Enzymol., 7, 35-39 (1995). The NOrelease profiles of two representative silica nanoparticles (10 and 17mol % of AHAP3 and AEAP3, respectively, balance TEOS) are compared inFIG. 22. Notably, the NO “payload” and release rates were significantlyaffected by both the concentration and chemical structure of the amineligands used to prepare the silica nanoparticles. Of the fouraminoalkoxysilane systems studied (e.g., AEAP3, AHAP3, AEMP3, and DET3),AEAP3 silica released the largest overall amount of NO. Increasing themol % of AEAP3 from 10 to 17 mol % led to a corresponding increase inboth t[NO] and [NO]_(m) (145 to 600 nmol/mg and 14 to 140 ppb/mg,respectively). However, both the t_(1/2) and t_(m) decreased withincreasing aminoalkoxysilane concentration (12 to 3.4 h and 8.0 to 2.1 hfor 10 to 17 mol % AEAP3, respectively). Significant levels of NOcontinued to be released for up to 30 h, albeit at a lesser rate forboth 10 and 17 mol % AEAP3.

One possibility is that such NO release behavior can be attributed tothe size of the particle. The diameter and surface areas of calculatedfor some of the presently described particles are shown below in Table5. As the diameter of the particle decreases for a givenaminoalkoxysilane (by increasing the aminoalkoxysilane concentration), asmaller water diffusion distance to interior NO donor ligands isexpected. As such, the NO release becomes more rapid sinceN-diazeniumdiolate decomposition to NO is a function of water uptake.Notably, the NO release properties of these silica particles deviatesfrom those of small molecule N-diazeniumdiolates and NO-releasing silicaprepared by surface grafting. Indeed, t_(1/2) of the AHAP3 silica wasfound to be 0.85 h, longer than t_(1/2) of 0.05 and 0.72 h for analoguessmall molecule DMHD/NO and the surface-grafted silica NO donors preparedwith N-(6-aminohexyl)-3-aminopropyltrimethoxysilane (see Zhang, H., etal., J. Am. Chem. Soc., 125, 5015-5024 (2003)), respectively, preparedusing similar amine precursors (i.e., aminohexylamino ligands) Likewise,t_(v2) of the AEAP3-based silica particles prepared via a “one-pot”synthesis was 3.4-12 h, while t_(1/2) of the surface grafted AEAP3silica (designated 2N[2] in Zhang, H., et al.) was reported as 2.4 h.See Zhang, H., et al., J. Am. Chem. Soc., 125, 5015-5024 (2003).

TABLE 5 Diameters and Surface Areas of of Silica Particles Preparedbased on the Co-condensation of NO Donor Precursors. Ligand Type Mol%^(c) d_(AFM) (nm) A_(BET) (m2/g) AEP3 10 500 9 AEP3 13 210 10 AEP3 1792 14 AHAP3 10 20 17 None (control) 0 250 500

The effect of pH on the, NO release kinetics from the silica scaffoldswas also evaluated, as shown in FIG. 23. Consistent with the behavior ofsmall molecule N-diazeniumdiolates (see Davies K. M., et al., J. Am.Chem. Soc., 123, 5473-5481 (2001)), NO release was accelerated underacidic conditions (pH 3.3). Conversely, NO release was slowedconsiderably at elevated pH (9.5), consequently demonstrating a simplemethod for storing and transporting NO donor nanoparticles withoutsignificant deterioration of the N-diazeniumdiolate. The t[NO] wassimilar at all pH values, but the NO release kinetics were dramaticallyincreased at lower pH. A nine-fold increase in the maximum flux of NOreleased ([NOim) was observed at pH 3.3 compared to that at pH 7.4. Suchbehavior, combined with the pH dependent dissociation ofN-diazeniumdiolates seems to confirm that the dominant mechanism of NOrelease for the silica scaffolds is proton initiated.

Example 14 Use of CTAB as a Template in the Synthesis of NO-ReleasingMesoporous AEAP3-Silica Particles

Cetyltrimethyl ammonium bromide (CTAB) was used as a template in thesynthesis of mesoporous AEAP3-silica. The mesoporous silica was preparedas described above in Example 10, using 10 mol % AEAP3, Additionally,the AEAP3/TEOS silane solution contained 0.01 M of CTAB. Followingcondensation of the silane mixture, the particles were treated with 1MHCl in EtOH at 75° C. for 24 h to remove the CTAB. A schematicrepresentation showing a proposed cross-sectional view of a mesoporousNO-releasing silica particle Is shown in FIG. 24A.

The particles were analyzed using atomic force microscopy as describedin Example 11. See FIG. 24B. Nitric oxide release was also measured asdescribed in Example 11. The nitric oxide release (ppb) versus time (hr)for 3 mg of the mesoporous particles in PBS at 37° C. is shown in FIG.25.

Example 15 Synthesis of Silica Particles Based on Co-Condensation ofPre-Charged NO Donors

Although the NO release levels of the silica nanoparticles prepared fromthe co-condensation of NO donor precursors (which can also be referredto as a “post-synthesis charging” or simply “post-charging”) weresignificantly greater than small molecule diazeniumdiolates, theaminoalkoxysilane content used to prepare the nanoparticles was limitedto <20 mol % due to particle aggregation at higher aminosilaneconcentrations. Without being bound to any particular theory, it isbelieved that the aggregation can be attributed to interactions betweenthe amines and adjacent silanols and/or other amines via hydrogenbonding.

To increase the concentration of aminoalkoxysilanes, and thus the NOdonor content of the particles, an additional strategy for synthesizingthe silica nanoparticles of the presently disclosed subject matterinvolves the co-condensation of silanes containing diazeniumdiolates.Thus, in contrast to the method described in the Example 10, where thesilica nanoparticles were first synthesized and then pressurized(“charged”) with the NO gas necessary to form diazeniumdiolate NO donors(which can also be referred to as a “post-synthesis charging” or simply“post-charging”), the diazeniumdiolates can also be formed prior toco-condensation of the silica nanocomposites (i.e., “pre-charging”). SeeFIG. 5B.

Briefly, an aminoalkoxysilane solution was prepared by dissolving anappropriate amount of aminoalkoxysilane in a mixture of EtOH, MeOH, andNaOMe. The stirring solution was charged with NO (5 atm, 3 d) to formdiazeniumdiolate-modified aminoalkoxysilanes. Silane solutions were thenprepared by mixing TEOS with different ratios (10-75 mol %, balanceTEOS) of diazeniumdiolate-modified aminoalkoxysilane. The silanesolution was added into an EtOH solvent in the presence of an ammoniacatalyst. The resulting white precipitate was collected bycentrifugation, washed with EtOH, dried under ambient conditions, andstored in a sealed container at −20° C. until use. The results suggestthat the pre-charging strategy reduces aggregation because theaminoalkoxysilanes are first converted to diazeniumdiolates, therebyavoiding interaction of amine sites during particle formation. As such,the approach can be used to facilitate greater access of NaOMe and NO tothe amine precursors resulting in high yields of NO per mol ofaminoalkoxysilane precursor.

Example 16 NO-Release Properties of Particles Prepared fromCo-Condensation of Pre-Charged NO-Donors

The NO release properties of diazeniumdiolate-modified silicananoparticles prepared via the pre-charging approach described inExample 15 are summarized below in Table 6. Notably, both the total NOreleased (t[NO]) and the maximum amount of NO released ([NO]_(m)) wereincreased considerably compared to NO releasing-silica prepared by thepost-charging method at identical aminoalkoxysilane concentrations (SeeTable 4). For example, t[NO] and [NO]_(m) for 17 mol % AEAP3 wereincreased from 600 to 800 nmol/mg and 140 to 1200 ppb/mg, respectively.Without being bound to any particular theory, the elevated quantities ofNO release could be the result of a more homogeneous distribution of thediazeniumdiolate NO donors throughout the silica particle, as shown inFIG. 5B. More importantly, the pre-charging approach allows for anincrease in the aminoalkoxysilane content up to 45 mol % withoutaggregation, resulting in concomitant increases in t[NO] and [NO]_(m).

Methylaminopropyl-trimethoxysilane (MAP3), an aminoalkoxysilanecontaining a methyl-terminated secondary amine, was also used to prepareNO-releasing silica particles. By removing primary amines and thepotential for hydrogen bonding interactions, particles with MAP3aminoalkoxysilane concentrations up to 75 mol % and sizes ranging from80-400 nm can be synthesized depending on the solvent employed duringsynthesis. Additionally, increasing the mol % of MAP3 from 10 to 75 mol% led to a corresponding increase in the NO release characteristics(e.g., t[NO] increased from 1600 to 10200 nmol/mg). In addition, the NOrelease of MAP3-based silica particles was characterized by a greaterinitial NO release burst and shorter overall NO release half-life(33000-177000 ppb/mg and −5 min, respectively).

TABLE 6 NO Release Properties of Particles Prepared from Pre-Charged NODonors Ligand Mol t[NO] t½ [NO]_(m) t_(m) Type % (nmol/mg) (h) (ppb/mg)(h) AEP3 17 800 1.13 1200 0.12 AEP3 25 1200 1.45 1600 0.13 AEP3 35 15001.83 1400 0.13 AEP3 45 1700 2.17 1300 0.13 AHAP3 10 600 0.25 3400 0.05AHAP3 25 1600 0.30 9500 0.05 AHAP3. 35 2600 0.35 14500 0.08 AHAP3 453800 0.27 21700 0.13 MAP3 45 1600 0.08 33000 0.05 MAP3 55 2900 0.0860000 0.05 MAPS 65 5800 0.08 134000 0.05 MAPS 75 10200 0.07 177000 0.05

Example 17 Ovarian Cancer Cell Studies

To evaluate the tumoricidal potential of NO donor silica nanoparticles,the cytotoxicity of control and NO-releasing silica particles onimmortalized normal (T29) and cancer (A2780 and OVCAR-3) human ovarianepithelial cells was tested. MTT cell viability assays were performed asdescribed below. The3-(4,5-dimethylthiazol-2-yl-)-2,5-diphenyltetrazoliumbromide (MTT)proliferation assay was employed to determine the relative sensitivitiesof OVCAR-3 cells to PYRRO/NO. Cells were seeded in 6 replicates at1-5×10³ cells/well in 96-well microtiter plates, incubated overnight,and exposed to concentrations of NO donor and control pyrrolidinesolutions for 48 h. The NO-releasing medium was then removed andreplaced by MTT solution, upon which the cells were incubated for anadditional 4 h at 37° C. Following removal of the MTT, DMSO was added,and the absorption of the solution was measured at 560 nm using amicroplate reader.

As shown in FIG. 26, A2780 ovarian epithelial tumor cells were treatedwith varying doses of control and NO-releasing AHAP3 silica (0.013-1.0mg/mL) for 48 h. The viability of the A2780 cells was reduced uponexposure to NO-releasing AHAP3 silica at low doses, and theproliferation of A2780 cells was almost completely inhibited byNO-releasing AHAP3 silica at a dose of 0.50 mg/mL [minimum inhibitoryconcentration (MIC) at <5% survival; corresponding to 0.75 mM of NO]. Inaddition, the IC₅₀ dose (50% inhibitory concentration) of NO donor AHAP3silica was 0.02 mg/mL (0.03 mM NO). Notably, the inhibitoryconcentrations of the NO-releasing silica proved to be significantlylower than those of small molecule NO donors (e.g., MIC and IC₅₀ forPYRRO/NO were 4.4 and 2 mM NO, respectively).

Control silica nanoparticles also exhibited cytotoxic effects againstthe tumor cells (IC₅₀=0.12 mg/mL), albeit less than that of their NOreleasing counterparts. Without being bound to any particular theory,the undesirable cytotoxicity of control vehicles could be the result offree primary amines on the surface of the silica structures, as suchgroups have known cytotoxic properties. See Shi, X., et al., ColloidsSurf A, 272, 139-150 (2006). To reduce the cytotoxicity of control andNO-releasing nanoparticles with primary amines, the MAP3 aminosilane(containing only secondary amines) was employed to create morebiocompatible vehicles. As expected, the cytotoxicity of MAP3 controlsagainst the immortalized (T29) and tumor (A2780) cells was low, whereasNO-releasing MAP3 silica exhibited cytotoxicity against both T29 andA2780 cells. See FIG. 27. OVCAR-3 ovarian adenocarcinoma cells alsoshowed similar cytotoxic trends with increasing concentrations ofNO-releasing silica nanoparticles.

To investigate whether nanoparticle size affects cytotoxicity, twosilica nanoparticles (75 mol % MAP3, balance TEOS) of different particlesize (80 and 350 nm in diameter, hereafter referred to as s-MAP3 andL-MAP3, respectively) were synthesized. Silica diameter is easilytunable by varying the solvent system (e.g., alcohol) during the sol-gelprocess. See Harris, M. T. et al., J. Non-Cryst. Solids, 121, 397-403(1990). Increasing the molecular weight (MW) of the alcohol used duringsynthesis led to a corresponding increase in the particle size (e.g.,100% (v/v) ethanol and 50/50% (v/v) ethanol/butanol mixture were used toprepare s-MAP3 and L-MAP3, respectively). Cell viability was determinedby incubating T29 and A2780 with non NO-releasing control MAP3 particle(80 nm), s-MAP3, or L-MAP3 (0.4 mg/mL) for 48 h. See FIG. 28. Notably,the small diameter NO-releasing silica (s-MAP3) proved cytotoxic againstboth immortalized (T29) and cancer (A2780) cells (12±1.1 and 5±0.2%survival, respectively). In contrast, the larger NO-releasing silica(L-MAP3) was significantly more cytotoxic towards the tumor cells thanhealthy cells (37±2.0 versus 6±1.2% survival for T29 and A2780,respectively). The reduced toxicity of the larger NO delivery vehiclesagainst T29 cells represents a major step toward the development ofnanodevices capable of releasing tumoricidal concentrations of NO withminimal effect on healthy cells.

Example 18 Cellular Uptake

The cellular uptake of NO-releasing silica particles was studied usingconfocal fluorescence microscopy. Briefly, A2780 ovarian cancer cellswere plated to −20% confluency on MET-TEC® glass bottom microscopyplates and incubated overnight. Prior to imaging, the Incubation bufferwas discarded and replaced with Krebs-Henseleit imaging buffer [10 mMN-2-hydroxyethylpiperazine-N¹-2-ethanesulfonic acid (HEPES), pH 7.4]containing 100 nM tetramethylrhodamine dye (TMRM) to selectively stainthe mitochondria of the A2780 cancer cells (30 min incubation). TheNO-releasing silica nanoparticles were fluorescently labeled via theco-condensation of three silane precursors: fluorescein isothiocyanate(FITC)-modified aminopropyl-trimethoxysilane (APTMS), diazeniumdiolatedMAP3, and TEOS.

A Zeiss Laser Scanning Microscope (LSM 510; Carl Zeiss, Inc.,Oberkochen, Germany) was used to perform the fluorescence measurements.The red fluorescence of TMRM (helium-neon laser excitation at 543 nm)was monitored at 5 min and at 60 min to provide a map of theintracellular location of mitochondria and an outline of A2780 nuclei.See FIGS. 29C and 29D. A 100-μL aliquot of FITC-labeled NO-releasingMAP3 silica nanoparticles dissolved in the imaging buffer was addeddirectly to the cells on the stage of the microscope, yielding ananoparticle concentration of 0.1 mg/mL. Immediately, the greenfluorescence of the FITC-labeled silica nanoparticles (argon laserexcitation at 488 nm) was observed at 520 nm, resulting in the outlineof the A2780 cancer cells. Confocal images were collected at 5 minintervals to monitor the cellular uptake of the green fluorescentnanoparticles, FIG. 29A shows the cells after 5 min incubation with theFITC labeled MAPS silica particles. After 1 h, substantial intracellularaccumulation of nanoparticles was observed. See FIG. 29B. Additionally,the red fluorescence characteristic of mitochondrial viability wasabsent in a number of cells after 60 min (see FIG. 29D), and the cellsappeared to be shrinking in size, indicating cell death.

Example 19 Antimicrobial Activity Studies

Pseudomonas aeruginosa (ATCC #19143, from American Type CultureCollection Company, Manassas, Va., United States of America), agram-negative opportunistic pathogen was cultured in tryptic soy broth(TSB) to an optical density (OD_(λ=600 nm)) of approximately 0.2(corresponding to ˜1.0×10⁸ colony forming units [CFU]/mL, confirmed byserial dilutions). After pelleting the bacteria by centrifugation, theTSB culture media was discarded and the bacteria were resuspended insterile phosphate buffered saline (PBS, pH 7.4). The concentration ofbacteria was adjusted to 10³ CFU/mL by serial 10-fold dilutions in PBS.Portions of this bacterial suspension (200 μL) were dispensed intosterile micropipette vials, and 200 μL of either NO-releasing 45 mol %AEAP3 silica nanoparticles (1 mg/mL), control (non NO-releasing) AEAP3silica nanoparticles (1 mg/mL) or sterile PBS (blank) were added to eachvial. After incubation at 37° C. for 1 h, 100 μL of each suspension wasplated onto tryptic soy agar nutrient plates, which were incubated at37° C. overnight. The following day, colonies of bacteria that formed oneach plate were counted and photographs of representative nutrientplates were taken. As shown in FIG. 30, nitric oxide release from silicananoparticles resulted in a drastic reduction in the number of viablebacteria cells (FIG. 30C), as compared to blank (FIG. 30A) and control(non NO-releasing) silica nanoparticles (FIG. 30B). Quantitatively,approximately the same number (˜360) of colonies formed on the platesrepresenting blank and control suspensions. Only 9 colonies formed fromthe suspension to which NO-releasing silica nanoparticles were added.This represents a 98% decrease in the number of viable bacteria cellsbetween suspensions to which NO-releasing nanoparticles were addedcompared to blank and control suspensions.

To more quantitatively evaluate antimicrobial activity of NO-releasingsilica nanoparticles, the concentration of bacteria was adjusted to 103CPU/mL by serial dilutions in PBS and cultures were exposed either tocontrol (non NO-releasing) silica nanoparticles, NO-releasing silicananoparticles, or sterile PBS (blank). After incubation for 1 h at 37°C., 100 pL of each suspension was plated onto tryptic soy agar nutrientplates and were incubated overnight. As shown in FIG. 31, NO releasefrom silica nanoparticles resulted in a drastic reduction in the numberof viable bacteria cells. At a concentration of 2 mg/mL, NO-releasingnanoparticles had a significant increase in bactericidal activity overcontrols (p=9.5×10⁻⁴). The quantity of NO released during the 1 hincubation period was approximately 1 μmol of NO as determined viachemiluminescence. The silica nanoparticles presented herein thusexhibit in vitro bactericidal activity and represent a vehicle fordelivering concentrations of NO for killing microorganisms relevant toinfected wounds.

Example 20 Synthesis of NO-Releasing Magnetic Silica Nanoparticles

Magnetic NO-releasing silica nanoparticles were prepared according tothe synthesis shown in FIG. 32. In short, the method of Example 10 wasadapted by the inclusion of magnetite (Fe₃O₄) particles having diametersof between about 20 nm and 30 nm in a solution containing TEOS andeither 10 mol % AHAP3 or 17 mol % AEAP3. Upon co-condensation of thesilanes, the magnetite particles were covered with a shell of silica.The particles were then subjected to NO to form diazeniumdiolates.

Atomic force microscopy (AFM) images of the magnetite/silica-AHAP3particles are shown in FIG. 33. The diameter of the particles wasmeasured as 85±11 nm. The NO-release profiles of the particles are shownin FIG. 34. Experiments with PBS solutions containing themagnetite/silica particles indicate that the application of a magnet cancontrol particle movement.

Example 21 Polyurethane Films Containing NO-Releasing SilicaNanoparticles

NO-releasing silica nanoparticles were incorporated into polyurethanefilms prepared by adding between about 3 mg to about 18 mg ofNO-releasing particle to polymer percursor solutions containing 10 mg of1:1 (w/w) TECOFLEX® polyurethane (TPU)/hydrophilic polyurethane (HPU) in500 1.1.L of THE and ethanol prior to polymerization.

The film prepared by adding 6 mg of nanoparticle to the polymerprecursor solution was tested to determine its ability to resistbacterial adhesion as previously described. See Marxer, S. M., et al.,Chem. Mater., 15, 4193-4199 (2003). The films were pre-treated toinitiate steady NO release and subsequently immersed in a cellsuspension containing Pseudomonas aeruginosa (ATCC #19143, from AmericanType Culture Collection Company, Manassas, Va., United States ofAmerica), at 37° C. for 30 min. The film surface was then rinsed withwater and fixed in a 2% glutaraldehyde solution for 15 min. images ofthe surfaces were obtained using phase contrast microscopy using a ZeissAxiovert 200 inverted microscope (Carl Ziess Optical, Chester, Va.,United States of America). Phase contrast optical micrographs of controlfilms and the NO-releasing particle-containing film are shown in FIGS.35A and 35B.

Example 22 Glucose Sensor with an NO-Releasing Layer

Glucose oxidase-based glucose biosensors can detect blood glucosethrough the electrooxidation of hydrogen peroxide generated by theglucose oxidase (GOx)-catalyzed reaction of glucose and oxygen. As shownschematically in FIG. 36, a glucose sensor was prepared having aNO-releasing layer, Sensor 3600 provides four layers stacked upon a Ptelectrode 3602 inner-most layer 3604 was formed from the condensation ofa solution containing 25 μL MTMOS, 6 mg of glucose oxidase (GOx), 100 μLEtOH, and 50 μL H₂O. Covering GOx layer 3604 is a protective layer 3606prepared from the polymerization of a 1:1 (w/w) mixture of hydrophobicTECOFLEX® polyurethane (TPU) and hydrophilic polyurethane (HPU)precursors (i.e, a TPU/HPU blend). NO-releasing layer 3608 was preparedfrom the polymerization of a solution containing 10 mg TPU/HPU and 6 mgof diazeniumdiolate modified silica nanoparticles in 500 μL of THF/EtOH.NO-releasing layer 3608 is further surmounted with a TPU/HPU barrierlayer 3610 prepared from a mixture of 10 mg TPU/HPU in 500 μL THF/EtOH.

Continuing with FIG. 36, the inset shows the interactions at theinterface of NO-releasing layer 3608 and outer protecting layer 3610,wherein NO-releasing silica particles 3620 having diazeniumdiolategroups 3622 release nitric oxide 3624 while glucose molecules 3626 areabsorbed into NO-releasing layer 3608 on their way to GOx-containinglayer 3604.

To evaluate the response of glucose sensor having NO-releasing layers,two control electrodes were also prepared: a control sensor having onlya protecting layer and a GOx layer, and a sensor containing all fourlayers only prepared with silica nanoparticles that did not containNO-donors. The sensitivity of the various sensors was evaluated in PBS(0.05 M, pH 7.4) using an applied potential of +7 V vs. Ag/AgCl. Thesensitivity of the control, two-layer sensor was determined as 54.5nA/mM (r=0.9980), that of the four-layer sensor with non NO-releasingsilica nanoparticles was 61.3 nA/mM (r=0.9938) and that of the sensorwith the NO-releasing layer was 57.9 nA/mM (r=0.9989). These resultsindicate that the NO-release does not interfere with GOx-based glucosesensing.

REFERENCES

The references listed below as well as all references cited in thespecification are incorporated herein by reference to the extent thatthey supplement, explain, provide a background for or teach methodology,techniques and/or compositions employed herein. All cited patents andpublications referred to in this application are herein expresslyincorporated by reference.

-   Albert, K., and Bayer, E. J., J. Chromatogr., 544, 345-370 (1991).-   Albina, J. E., and Reichner, J. S., Canc., Metas. Rev., 17, 19-53    (1998).-   Anwander, R., et al., Stud. Surf. Sci. Catal., 117, 135-142 (1998).-   Baker, J. R., Jr., Biomacromolecules, 5, 2269-2274 (2004).-   Beckman, J. S., and Conger, K. A., Methods Companion Methods    Enzymol., 7, 35-39 (1995).-   Brannon-Peppas, L. and Blanchette, J. O., Advanced Drug Delivery    Reviews, 56, 1649-1659 (2004).-   Bruch, M. D., and Fatunmbi, H O., J. Chromatogr, A., 1021, 61-71    (2003).-   Brust, M., J. of the Chem. Soc., Chem. Comm., 801-802 (1994).-   Capala, J., et al., Bioconjugate Chem., 7(1), 7-15 (1996).-   Cobbs, C. S., et al., Cancer Res., 55, 727-730 (1995).-   Davies, K. M., et al., J. Am. Chem. Soc., 123, 5473-5481 (2001).-   Diodati, J. G., et al., Thrombosis and Haemostasis, 70, 654-658    (1993).-   Feldheim, D. L. and Foss, C. A., eds, Metal Nanoparticles—Synthesis    Characterization, and Applications. Marcel Dekker, Inc: New York, p.    360 (2000).-   Freireich et al., Cancer Chemother Rep. 50, 219-244 (1966).-   Frost, M. C., et al., Biomaterials, 26, 1685-1693 (2005).-   Harris, M. T., et al., J. Non-Cryst. Solids, 121, 397-403 (1990).-   Hatton, B., et al., Acc. Chem. Res., 38, 305-312 (2005).-   Hostetler, M. I., et al., Langmuir, 15, 3782-3789 (1999).-   Hostetler, M. I., et al., Langmuir, 14, 17-30 (1998).-   Hrabie, J. A., et al., J. Org. Chem., 58, 1472-1476 (1993).-   Hrabie, J. A. and Keefer, L. K., Chem. Rev., 102, 1135-1154 (2002).-   Huh, S., et al., Chem. Mater., 15, 4247-4256 (2003).-   Ignarro, L J., Nitric Oxide: Biology and Pathobiology; Academic    Press: San Diego (2000).-   Ignarro, L. J. et al., Proc. Natl. Acad. Sci., U.S.A., 84, 9265-9269    (1987).-   Jenkins, D. C., et al., Proc. Natl. Acad. Sci., U.S.A., 92,    4392-4396 (1995).-   Keefer, L. K., Annu. Rev. Pharmacol. Toxicol., 43, 585-607 (2003).-   Keefer, L. K., Chemtech, 28, 30-35 (1998).-   Lai, C.-Y., et al., J. Am. Chem. Soc., 125, 4451-4459 (2003).-   Lim, M. H., and Stein, A., Chem. Mater., 11, 3285-3295 (1999).-   Lin, H.-P., and Mou, C-Y., Acc. Chem. Res., 35, 927-935 (2002).-   Marletta, M. A., et al., BioFactors, 2, 219-225 (1990).-   Marxer, S. M., et al., Chem. Mater., 15, 4193-4199 (2003).-   Munoz, B., at al., Chem. Mater., 15, 500-503 (2003).-   Nablo, B. J., et al., J. Am, Chem. Soc., 123, 9712-9713 (2001).-   Napoli, C. and Ignarro, L. J., Annu. Rev. Pharmacol. Toxicol., 43,    97-123 (2003).-   Penault-Llorca, F., et al., Int. J. Cancer, 61(2), 170-176 (1995).-   Press, M. F., et al., Oncogene 5(7), 953-962 (1990).-   Radomski, M. W., et al., Br. J. of Pharmacology, 101, 145-749    (1992).-   Radu, D. R., et al., J. Am. Chem. Soc., 126, 1640-1641 (2004).-   Roy, I., et al., Proc. Natl. Acad. Sci, U.S.A., 102, 279-284 (2005).-   Sayari, A., and Hamoudi, S., Chem. Mater., 13, 3151-3168 (2001).-   Shi, X., et al., Colloids Surf A., 272, 139-150 (2006).-   Stein, A., et al., Adv. Mater., 12, 1403-1419 (2000).-   Thomsen, L. L., et al., Br, J. Cancer., 72, 41-44 (1995).-   Trewyn, B. G., et al., Nano. Lett., 4, 2139-2143 (2004).-   Troughton, B. B., et al., Langmuir, 4, 365-385 (1988).-   Wang, P. G., et al., Nitric Oxide Donors: For Pharmaceutical and    Biological Applications; Wiley-VCH: Weinheim, Germany (2005).-   Wang, P. G., et al., Chem. Rev., 102, 1091-1134 (2002).-   Wiener, E. C. et al., Invest. Radiol., 32 (12), 748-754 (1997).-   Wiener, E. C., et al., Magn. Reson. Med. 31(1), 1-8 (1994).-   Yoshitake, H., New. J. Chem., 29, 1107-1117 (2005).-   Zhang, H., et al., J. Am. Chem. Soc., 125, 5015-5024 (2003).-   Zhou, Z., and Meyerhoff, M. E., Biomacromolecules, 6, 780-789    (2005).

It will be understood that various details of the presently disclosedsubject matter can be changed without departing from the scope of thepresently disclosed subject matter. Furthermore, the foregoingdescription is for the purpose of illustration only, and not for thepurpose of limitation.

That which is claimed:
 1. A nitric oxide-releasing particle comprising:a co-condensed silica network comprising methylaminopropyltrimethoxysilane (MAP3) and tetra methyl orthosilicate (TMOS), whereinthe MAP3 is distributed throughout the nitric oxide-releasing particle,and wherein the MAP3 comprises N-diazeniumdiolate functionalized MAP3.2. The nitric oxide-releasing particle of claim 1, wherein the MAP3 ispresent in a mol % of 10% to 75%.
 3. The nitric oxide-releasing particleof claim 1, wherein the MAP3 is present in a mol % of 1% to 90%.
 4. Thenitric oxide-releasing particle of claim 1, wherein the MAP3 is presentin a mol % of 45% to 55%.
 5. The nitric oxide-releasing particle ofclaim 4, wherein the MAP3 is present in a mol % of about 50%.
 6. Thenitric oxide-releasing particle of claim 1, wherein theN-diazeniumdiolate functionalized MAP3 comprises a sodium cation.
 7. Thenitric oxide-releasing particle of claim 1, wherein theN-diazeniumdiolate functionalized MAP3 comprises a potassium cation. 8.The nitric oxide-releasing particle of claim 1, wherein theN-diazeniumdiolate functionalized MAP3 comprises a lithium cation. 9.The nitric oxide-releasing particle of claim 3, wherein the TMOS ispresent in a mol % of 10% to 99%.